JP4825426B2 - Dark field illuminator for biological microscopes - Google Patents

Description

  The present invention relates to a biological microscope having a low magnification to a high magnification, which can perform bright field observation and dark field observation with a wide field of view range and sufficient brightness, and a dark field illumination device used therefor.

  In the fields of medicine, biology, and engineering, a biological microscope is placed in a dark field state and a sample such as a cell is observed. A structural diagram of a conventional biological microscope is shown in FIG. Most of the light sources 1 use tungsten light bulbs or halogen light bulbs, but biological microscopes that use a plurality of high-intensity light-emitting diodes arranged on a plane as a light source are also commercially available. As shown in FIG. 22, the dark field illumination device used in this biological microscope condenses the light beam emitted from the light source 1 built in the biological microscope with the condenser lens 2 and reflects the collected light beam to the reflecting mirror. 3 to change the direction, the central part of the light beam is shielded by the disk-shaped shielding object MS of the dark field condenser AL, and the cylindrical light beam in the peripheral part is further condensed by a specially shaped mirror ML to obtain a funnel. In this way, the sample is dark-field illuminated. By using a funnel-shaped light beam, the irradiation angle and the irradiation area are uniquely limited, and it has been difficult to obtain a wide range of dark field illumination.

  For example, FIG. 23 is a photomicrograph of a dark field image of a brain tissue of an animal using a conventional dark field illumination device of a commercially available biological microscope. The micrographs A to D in FIG. 23 were respectively taken by changing the magnification of the objective lens 6 to 20 ×, 10 ×, 4 ×, and 2 × in order of low magnification. At A with a magnification of 20 times, the entire visual field can be observed in a dark field, but at B with a magnification of 10 times, the surroundings do not appear dark due to insufficient light quantity. Comparing this 10x magnification B with 4x magnification C and 2x magnification D, the lower the magnification, the narrower the illumination range and the worse the amount of light, both of which are illuminated only in the center. I understand that. E and F in FIG. 23 show that the magnification of the objective lens is changed to 10 × magnification and 4 × magnification and the condenser lens is moved up and down to widen the irradiation range. ing. In any case, when the magnification is 10 times or less, the dark field illumination is insufficient. Although the field of view viewed from the microscope eyepiece 7 with the naked eye is circular, all of the images in FIG. 23 are missing images due to the restrictions of the camera photographing system, but do not affect the evaluation. .

As other conventional technologies, the light is transmitted through a dark field illumination device using a light bulb and a mirror incorporated in a stereomicroscope stage as shown in FIG. 24 and a light emitting diode disclosed in Japanese Patent Application Laid-Open No. 2003-75725 (see Patent Document 1). There is a stereo microscope that is built in an illumination stand and enables dark field observation, but there is no stage mechanism for moving the sample, the observation magnification is low, and the image quality is inferior, so it cannot be used for a biological microscope. .
JP 2003-75725 A

  The stereomicroscope has a magnification of the objective lens of about 0.5 to 5 times, and the distance from the objective lens to the sample is much longer than that of the biological microscope. In other words, the sine of half of the numerical aperture NA (the open angle (the angle at which the effective diameter of the objective lens is sandwiched at the object focal point on the optical axis of the objective lens) in the refractive index of the medium between the objective lens and the subject). (Multiplied value) is also smaller than that of a biological microscope, and when the magnification is the same, the resolution and depth of focus are completely different. Therefore, it is obvious that the dark field illumination device of the stereomicroscope cannot be applied structurally even if it is applied to the field of the biological microscope.

  The technique disclosed in Japanese Patent Application Laid-Open No. 2003-75725 is described as an angle larger than NA or an angle smaller than NA in the published patent gazette, but this is an incorrect description and is not “NA” described in the publication. If it is not correctly interpreted as “an opening angle defining NA”, it does not fit logically. Assembling the device without noticing a logical mistake in this way illuminates areas other than those that require night vision due to the difference in irradiation angle, which is not useful for observation. Therefore, this stereomicroscope technique cannot be applied as a dark field device for a biological microscope without the idea of illuminating dark field with strict definition of resolution and depth of focus.

  In the stereomicroscope, as shown in FIG. 24, a glass plate is arranged on a sample stage for locating the sample. Therefore, a glass plate for a sample is provided between the light source 1 and the sample 5 in transmission illumination or dark field illumination. There are two surfaces of the glass plate for sample mounting. In the case of dark field illumination, dust or dirt adhering to the surface of the glass plate due to static electricity or the like is emphasized, resulting in obstruction of observation or image degradation. The stereomicroscope has a deep depth of focus, so it will also focus on these dusts and scratches on the glass plate. FIG. 25 shows an observation image of the stereomicroscope under dark field illumination. FIG. 25A shows a sample as a specimen. In FIG. 25B, the sample is retracted, and a lot of dust shines on the inner and outer surfaces of the glass plate on which the sample is placed, which hinders observation.

  Furthermore, in the stereomicroscope, the distance (WD) from the objective lens to the observation sample is several centimeters to several tens of centimeters, which is much longer than that of the biological microscope. Conversely, the diameter of the objective lens for this WD is generally smaller than that of a biological microscope. Therefore, the angle of oblique illumination necessary for dark field illumination is also small with respect to the optical axis of the objective lens, but the low magnification objective lens of the biological microscope displayed the necessary number of fields (the diameter of the visible intermediate image in millimeters). In order to obtain a number (usually 20 to 25), even if the aperture of the objective lens is enlarged and oblique illumination is performed at an angle larger than the aperture angle that defines the numerical aperture of the lens, the light hits the lens. Sometimes, simply illuminating at an angle larger than the angle that defines NA does not provide clean dark field illumination.

  For example, an objective lens of a biological microscope as shown in FIG. 26 has a WD of 3.2 mm, a diameter of 20 mm, and an NA of 0.04. The angle defining the NA of this lens is 2.29 degrees, whereas the half of the opening angle created by the lens aperture is 72.2 degrees. When the sample is irradiated at the same angle as the dark field illumination of a stereomicroscope, the light enters the inside of the objective lens and generates flare, as shown on the right side, even though the angle defining the NA is larger. Or light reflected on the lens surface or inside is easily reflected on the glass of the sample (preparation) and enters the lens again. From the above, even if a dark field illumination device of a stereomicroscope is simply incorporated in a biological microscope as it is, it cannot serve as a dark field device.

  FIG. 27 shows how the dark field illumination range changes with respect to the entire visual field range that can be observed from the eyepiece when the magnification of the objective lens is changed for the biological microscope and the stereomicroscope, and changes in the characteristics of both. FIG. The dark field illumination range of the conventional biological microscope and stereomicroscope is a hatched area in the figure. In the range from magnification 1 to magnification 40, the biological microscope has a magnification that can dark-field illuminate the entire range from 20 to 40 times, and the illumination range is narrowed at low magnification. On the other hand, in the stereoscopic microscope, the dark field illumination range covers almost the same range as the entire visual field range from about 1 to 4 times magnification, but the magnification range covers from about 1 to 4 times. However, at a magnification of 4 to 10 times, the entire visual field range is not covered with dark field illumination by either a biological microscope or a stereomicroscope.

  Most conventional biological microscopes use a bright-field light source built in the microscope, and the objective lens magnification is wide enough at a low magnification such as 10 times, 4 times, and 2 times with sufficient brightness. The visual field range could not be illuminated with dark field, and the entire visual field could not be observed with dark field illumination from high magnification to low magnification. In addition, the conventional dark-field illumination device of a biological microscope uses a dark-field condenser lens, so that when the sample is illuminated for a long time, the dark-field condenser lens heats the sample with concentrated light heat, so cultured cells that dislike temperature rise The dark field illumination could not be performed.

  In addition, the stereomicroscope can perform dark field illumination at a low magnification, but as shown in FIG. 27, the dark field illumination range is limited to a limited magnification, and observation by dark field illumination at a high magnification is not possible. I could not do it. In other words, the stereo microscope only covers 1 to 4 times magnification, and the biological microscope can illuminate the entire range with dark field illumination at 20 to 40 times, and the illumination range becomes narrower at low magnification. Both were short and long, and the range from low to high magnification could not be observed continuously with dark field illumination. Observing a biological microscope and a stereomicroscope by exchanging them took a long time to set, was cumbersome, hindered the progress of research observation, and the workability was poor.

  Furthermore, with conventional biological microscopes, it has been impossible to switch to dark field observation instantaneously or perform simultaneous use of bright field and dark field illumination while performing normal bright field observation. When observing the structure of cells, light transmittance varies depending on the structure that easily reflects light irregularly, granular labeled substances, membranes, blood vessel walls, and staining conditions. Although it was necessary to use them properly, in the conventional biological microscope, the bright and dark field condenser lens and the dark field condenser lens have different height positions when observing, so when removing and replacing this, remove the screws one by one, It was necessary to tighten the screw or adjust the height position, and each time required a minute unit to obstruct the flow of observation.

  In view of such problems of the conventional biological microscope, the present invention can illuminate the entire visual field range from low magnification to high magnification, and only bright field illumination or only dark field illumination depending on the observation situation. It is possible to switch to both bright field illumination and dark field illumination at the same time, and easily switch to transmissive dark field illumination or epi-illumination dark field illumination depending on the object to be observed. An object of the present invention is to provide a biological microscope capable of performing the above and a dark field illumination device used therefor.

First aspect of the present invention, a light flux emitted from the plurality of semiconductor light sources arranged on a concentric circle, by irradiating the sample surface placed perpendicular to the optical axis of the observation light, a dark field observation of the sample A dark-field illumination device for use in a biological microscope that enables a hollow base, a hollow ring-shaped frame that is screwed to the base, and a fixing that is erected on the circumference of the ring-shaped frame on one end side rotating washer and a light beam toward the sample surface with electrically insulated to the other end of the rotary washers which have a stop hole in which the pin enters rotates as a fulcrum fixed pin with the rotation of the annular frame A plurality of semiconductor light sources arranged so as to irradiate each of the light sources, and the rotating washer is rotated with the rotation of the ring-shaped frame to change the diameter of the irradiation region. It is a field illumination device.

  Since the present invention is configured as described above, the following effects can be obtained.

  Since it is a biological microscope and a dark field illuminating device having a dark field illuminating device having a light illuminating angle varying means for varying the illuminating angle of the light beam radiated toward the sample from the oblique direction of the observation optical axis, A wide range of regions that could not be observed can be observed in the entire visual field range from low magnification to high magnification. In the dark field observation, the irradiation angle of the irradiation light beam can be finely changed, so that it is possible to adjust to a position where the light source itself or flare does not appear in the field of view, and an appropriate dark field illumination effect can be obtained. In addition, according to the necessity of observation, only bright-field illumination or only dark-field illumination can be switched, or both can be switched or returned as appropriate, or both bright-field illumination and dark-field illumination can be observed simultaneously.

  Also, by using a semiconductor light source, unlike conventional biological microscopes that place a dark field condenser that concentrates light heat from the bottom of the sample even under long-time illumination, it does not become a heat source, thus preventing the temperature of the sample from rising and cultivating cultured cells. Is also suitable. Furthermore, since the base and the ring-shaped frame are hollow and the objective lens can be arranged in this hollow portion, incident dark field illumination is also possible depending on the observation object.

  The dark field illumination device is arranged concentrically at intervals along a hollow ring-shaped frame provided in contact with the hollow base, and irradiates the sample surface placed perpendicular to the observation optical axis. The structure of the sample surface is simple because the irradiation angle of the light beam applied to the sample from a plurality of semiconductor light sources that respectively generate the light changes by rotating a ring-shaped frame screwed to the base. The illumination angle and illumination range of the light beam emitted toward the camera can be changed smoothly and finely, and the dark field illumination range is optimized by a simple operation of rotating the ring frame with one hand while observing the sample from the eyepiece. Can be adjusted.

  In a dark field illumination device in which a plurality of semiconductor light sources are arranged concentrically in a zigzag pattern along the inner wall of the ring frame or base, the sample can be irradiated with light smoothly over a wide range, depending on the condition of the sample The semiconductor light source can be selectively turned on and off to change the irradiation region of the light flux.

  In a dark field illumination device provided with a cylindrical light shielding cover that guides a light beam emitted from a semiconductor light source in one direction, extraneous light from the outside is prevented from entering the objective lens, and flare of an observation image is prevented. The occurrence of is reduced.

  In a dark field illumination device in which each of a plurality of semiconductor light sources is provided on a rotating washer and the direction of the light beam irradiated by rotating the rotating washer is rotated as the ring frame rotates, the dimensions of the existing biological microscope Even if the diameter of the annular frame is fixed depending on the shape, the diameter of the illumination area formed by the light beam emitted from the semiconductor light source mounted on the annular frame can be freely changed by rotating the rotating washer. Can be widened, narrowed, or adjusted so as to be in an optimum position according to the sample.

  In the dark field illumination device in which the reflecting surface for turning the direction of the light beam irradiated from the plurality of semiconductor light sources is provided inside the ring-shaped frame, there is no movable part in the way of the wiring to the semiconductor light source. High reliability and long life.

An embodiment of the present invention will be described based on an example with reference to the drawings.
FIG. 1 shows the appearance of the main part of the biological microscope. FIG. 1 shows a schematic diagram in which a dark field illumination device of the present invention is provided in place of the dark field condenser lens AL in the vicinity of a position where the dark field condenser lens AL of an existing biological microscope is provided, and a sample (not shown) In order to illuminate the sample 5 placed on the table, the sample 5 is arranged perpendicular to the observation optical axis L. Since the entire structure of the biological microscope is the same as that of the conventional biological microscope shown in FIGS. 21 and 22, the description thereof will be omitted, and the same members will be described using the same numbers in the following description, and different configurations will be described. The dark field illumination apparatus will be described in detail below.

  In the night vision apparatus according to the present invention, a hollow ring-shaped base 8 is fixed at a position corresponding to the attachment position of the dark field condenser lens AL of the biological microscope. The base 8 has a shape as shown in FIG. 2A and a cross-sectional shape as shown in FIG. 2B. A ring-shaped frame 9 is attached to the base 8. In the annular frame 9, light emitting diodes 10 serving as semiconductor light sources are arranged in an annular shape at regular intervals. That is, each light emitting diode 10 is erected on a small piece 11 made of a small square printed board, and this small piece 11 is pivotally supported on the upper end side so as to be rotatable about the inner upper position of the annular frame 9. 12 and extends toward the lower center of the ring-shaped frame 9. Each of the small pieces 11 rotates around a support shaft 12 perpendicular to a plane including the central axis L of the observation optical system and the optical axis of the light emitting diode 10, and irradiates from the light emitting diode 10 toward the center of the ring frame 9. It is attached so that the angle of the luminous flux (optical axis) is variable.

  A female screw 14 is cut below the inner wall surface of the annular frame 9. When the female screw 14 is screwed with the male screw 13 of the base 8 and the annular frame 9 is rotated, the axial direction of the annular frame 9 and the base 8 is increased. The relative position changes. Since the lower end of each small piece 11 to which each light emitting diode 10 is attached is in contact with the upper surface of the base 8, when the ring-shaped frame 9 is rotated, the tangent line of this lower end acts as an action point, and the support shaft 12 on the upper end side of the small piece 11. As a fulcrum, each small piece 11 rotates and the elevation angle of the light emitting diode 10 changes smoothly, and accordingly, the irradiation range gradually changes smoothly. Therefore, by rotating the ring-shaped frame 9 while viewing the sample 5 from the eyepiece 7, the dark field state is changed minutely in units of micrometers and millimeters, and the sample 5 to be observed is observed with an optimum image quality or photographed. Is possible.

  The light emitting diode 10 of the dark field illumination device is powered by a battery (not shown) housed inside the annular frame 9 and is turned on and off by a switch (not shown). In the case where the battery is not housed in the ring-shaped frame 9, power may be supplied by an appropriate means such as an electric wire or electromagnetic coupling.

  FIG. 3 shows a photomicrograph comparing the dark field illumination device according to the present invention with an observation image of a sample using a conventional dark field condenser. As shown in FIG. 3A, according to the dark field illumination device of the present invention, even when the dark field observation is performed with the objective lens 2, the dark field illumination can be performed over the entire field of view, and the entire region can be observed. If the photograph is taken from the three eyeglass tubes, the photographing range becomes small, so that it can be used from the objective lens 1 ×. On the other hand, as shown in FIG. 3B, in the conventional biological microscope using the dark field condenser, in the case of the dark field observation of 1 to 2 times the objective lens, only the central portion can be observed and the surrounding is dark. I can't see. The micrograph in FIG. 3 corresponds to the diagram in FIG. 27 showing the relationship between the magnification and the dark field illumination range.

  In FIG. 2, the plurality of light emitting diodes 10 are arranged at substantially the same height position inside the hollow ring-shaped frame 9, and the irradiation angle is changed on the spot. In Example 1 of FIG. 4, light is emitted uniformly over a wide range, and therefore, ultrahigh-brightness light emitting diodes 10a and 10b, which are semiconductor light sources, are arranged in a staggered manner inside the annular frame 9 at regular intervals. This is an example. The light emitting diode 10a exclusively illuminates the central part of the observation visual field, and the light emitting diode 10b exclusively illuminates the peripheral part. By arranging them alternately in this way, a wide area can be irradiated obliquely as shown in the shadowed portion of the schematic diagram of FIG. In this case, by turning on the light emitting diode 10a or turning on the light emitting diode 10b with a switch (not shown), the irradiation angle of the light beam applied to the sample from the oblique direction of the observation optical axis can be greatly changed. The function of the irradiation light angle varying means can be achieved. In this case, the irradiation light angle is slightly changed by the vertical movement of the annular frame 9 caused by the rotation of the annular frame 9. It is possible to substantially change the irradiation angle of the light beam by arranging the light emitting diodes 10a and 10b not only in two layers but also in multiple layers, multiple layers, densely, or selectively lighting.

FIG. 6 shows an example in which the high-intensity light emitting diodes 10 as semiconductor light sources are arranged in a staggered manner, and the irradiation angle of the light beam emitted from the light emitting diodes 10 changes minutely as the ring-shaped frame 9 rotates. is there. In this embodiment, light emitting diodes 10a and 10b are attached to square pieces 11a and 11b each having a support shaft 12 perpendicular to a plane including the central axis L of the observation optical system and the optical axis of the light emitting diode 10, respectively. The small piece 11 a and the small piece 11 b are fitted in a recess in the inner wall of the ring-shaped frame 9 so as to rotate about the support shaft 12. Light emitting diodes 10a and 10b are provided on the surfaces of the small piece 11a and the small piece 11b so that the optical axes are vertical. The difference between the small piece 11a and the small piece 11b is that the light emitting diodes 10a and 10b are arranged in a staggered manner inside the ring-shaped frame 9, so that the mounting position of the light emitting diode 10a of the small piece 11a and the mounting position of the light emitting diode 10b of the small piece 11b are different. Is slightly different in position.

  The irradiation angle of the light beam emitted from the light emitting diodes of the annular frame 9 can be adjusted to an arbitrary angle by rotating the annular frame 9. A female screw 14 is cut on the lower inner peripheral surface of the ring-shaped frame 9. When the female screw 14 is screwed into the male screw 13 of the base 8 and the ring-shaped frame 9 is rotated, the ring-shaped frame 9 is positioned in the axial direction. Can be moved so that the irradiation angle of the luminous flux becomes optimum. By rotating the ring-shaped frame 9 in the axial direction with respect to the base 8, the angles of the small pieces 11a and 11b that are in contact with the upper surface of the base 8 change relatively, and the small pieces 11a with the tangent in contact as the point of action. , 11b rotate, the elevation angle of the light emitting diodes 10a, 10b changes minutely, and the irradiation range changes.

  In the embodiment of FIG. 7, a cylindrical light-shielding cover 15 is provided on the bullet-type light emitting diode 10 so as to block the scattered light from the outside that interferes with the dark field illumination in the embodiment of FIG. 1 or the embodiment 2. It is a thing. This prevents extra light from entering the objective lens 6 and reduces the occurrence of flare in the observed image.

  In Example 3 of FIG. 7, the light beam emitted from the bullet-type light emitting diode 10 is directly used for dark field illumination. However, in Example 4 of FIG. 8, the reflecting plate 16 is attached to each light emitting diode 10, A reflecting plate 16 is placed in the optical path from the light emitting diode 10 to bend the optical axis. By doing this, the distance from the center of the device for dark field illumination to the light source portion is shortened, the outer periphery of the device is made compact, and the reflection plate 16 increases irradiation light and diffuses light to unnecessary portions. Can be prevented.

  Example 5 of FIG. 9 is an example using a surface-mount type light emitting diode 17 instead of using the bullet-type light emitting diode 10. In the fifth embodiment, one end of the small piece 11 is in contact with the groove inside the ring-shaped frame 9 in which a ring-shaped groove is formed on the inner side, and the luminous flux emitted from the light emitting diode 17 is arranged to be parallel to the plate surface of the small piece 11. Is done. The other end side of the small piece 11 is in contact with the base 8 and is spanned. The relative position between the base 8 and the ring-shaped frame 9 is changed by the rotation of the ring-shaped frame 9, and the range of light emitted from the light emitting diode 10 is smoothly changed. With this configuration, the size can be reduced. Further, by projecting the upper part of the ring-shaped frame 9 inward and processing the inner surface so that light is reflected to form a collar portion 18, there is no light shielding cover 15 like the bullet-type light emitting diode 10 of the third embodiment. However, it is possible to realize a dark field device that is hardly affected by external light. If comprised in this way, it will be possible to make it compact so that it can also be set in the gap between the bright field condenser and the microscope stage.

  An example in which the flexible substrate 19 is used for processing and wiring efficiency is shown in FIG. FIG. 10 shows an embodiment in which the light emitting diodes 10a and 10b are arranged in a zigzag pattern. The belt-like flexible substrate 19 is bent at the position of the dotted line, and this substrate is made cylindrical as shown in FIG. The cylindrical flexible substrate 19 is fitted into the ring-shaped frame 9. When the ring-shaped frame 9 is rotated, the relative position of the ring-shaped frame 9 and the base 8 moves, and the portion bent at the inner upper end of the ring-shaped frame 9 presses the flexible substrate 19. Since the bent portion has elasticity for closing, the bending angle of the flexible substrate 19 is changed by being pushed by the end face of the ring-shaped frame 9 as shown in FIGS. The illumination angle of the light source changes.

  Example 7 shows an example using a disk-shaped flexible substrate 20 as shown in FIG. As shown in FIG. 14, the light emitting diodes 10a and 10b are arranged radially. These light emitting diodes 10a and 10b are wired around the circle by printed wiring, and are bent at the position of the dotted line so as to have elasticity in the direction of opening at the root portion of the gear teeth. The bent disk-shaped flexible substrate 20 is mounted on a ring-shaped frame 9 that is screwed into the base 8 as shown in FIG. When the ring-shaped frame 9 is rotated, the ring-shaped frame 9 rotates with respect to the base 8, and the position in the axial direction changes relatively. Corresponding to this change, the contact point with the base 8 is changed by the movement of the ring-shaped frame 9 with the bent portion of the disk-shaped flexible substrate 20 as a fulcrum, so that the irradiation angle of the light flux from the light emitting diodes 10a and 10b, that is, the light source. change.

  In Example 8, as shown in FIG. 17A, the diameter of the illumination area formed by the light beam emitted from the light emitting diode 10 mounted on the annular frame 9 can be freely changed by the rotation of a plurality of rotating washers 21. It is something that can be done. Even if the diameter of the ring-shaped frame 9 is fixed, as shown by B in FIG. 17 and C in FIG. 17, the darkness in which the diameter of the illumination region is made substantially variable by changing the arrangement position of the light emitting diodes 10. It is an example of a field illumination device. The following treatment is applied to each of the base 8 and the ring-shaped frame 9 which are combined to form an internal / external double. A fixing pin 22 is erected on the base 8, and a fixing pin 23 is erected on the upper surface of the annular frame 9. A stop hole 24 for receiving the fixing pin 22 is provided at one end side of the rotary washer 21, and a slot-shaped long hole 25 is provided at the center. On the other end side of the rotary washer 21, the light emitting diode 10 is erected in a state where it is electrically insulated from the rotary washer 21. The light emitting diode 10 is provided with a cylindrical light shielding cover 15 similar to that of the third embodiment to enhance the directivity of the irradiated light beam. As shown by B in FIG. 17, a force for rotating the rotating washer 21 works with the fixing pin 22 as a fulcrum, and as shown by C in FIG. As a result, the entire irradiation area is widened or narrowed, so that it can be adjusted to an optimum position according to the sample 5.

  In the ninth embodiment, as shown in FIG. 18, the light emitting diode 10 is fixed to the upper end of the ring-shaped base 8, and the female screw 14 that engages with the male screw 13 carved on the outer periphery of the base 8 is carved. The ring-shaped frame 9 is configured not to mechanically contact the light emitting diode 10. That is, the light beam emitted from the light emitting diode 10 is reflected by the reflecting surface 26 provided on the inner upper side of the ring-shaped frame 9, and the traveling direction of the light is turned toward the center of the dark field illumination device, so that the sample is inclined. It is configured to illuminate from below. Since no current supply circuit for the light emitting diode 10 is mechanically damaged, no matter what the ring-shaped frame 9 is rotated for observation, the wiring of the light emitting diode 10 does not break.

  In each of the above embodiments, an example in which a night vision illumination device is provided on the opposite side of the observation optical system of the sample surface to be observed is shown in FIG. 19 because the dark field device according to the present invention has a hollow central portion. As shown, a bright field illumination system is incorporated in the hollow portion 41A of the annular frame 41 to enable epi-illumination, to switch between bright field illumination and dark field illumination alternately, bright field illumination and dark field illumination, Can be performed at the same time. FIG. 20 is a photomicrograph of observed cells obtained by staining animal brain cells with neutral red or DAB. FIG. 20A shows a micrograph of the sample observed only with bright-field illumination, with white holes indicating blood vessels and black particles indicating nerve cells. FIG. 20B shows a photomicrograph of bright field illumination and dark field illumination simultaneously performed on the same sample. C in FIG. 20 is a micrograph of the same sample observed only with dark field illumination, and blood vessels that can be observed in white with bright field illumination are conversely observed as black and neurons are white granules. Is missing.

  In the case of bright field illumination, the difference in light transmittance can be clearly observed, and the cell type can be distinguished by the degree of staining of the cells by staining. In the case of dark-field illumination, the structure of cells such as structures that easily diffuse light, granular labels, membranes, and blood vessel walls can be seen. When bright field illumination and dark field illumination are performed simultaneously, an intermediate image between the two can be obtained. In the case of bright field observation with an objective lens of 4 times or less, sufficient bright field illumination is possible with the built-in light source alone by removing the condenser lens. In addition, the entire field of view is not illuminated if the condenser lens is not removed in the observation with 1x objective. Therefore, it is convenient for the combined use of bright field and dark field.

  Therefore, by enabling illumination in three types of conditions, things that were not clearly understood when observed with dark field illumination can be revealed with bright field illumination, or the relative positional relationship on the tissue structure can be clearly understood. It contributes to research progress in the medical field and the bio field.

  Since the light source is composed of individual light sources arranged in a ring shape, the illumination direction can be biased by lighting a part of the light source. It is also possible to arrange light sources having different wavelengths and turn on the light sources for each type of wavelength. Since this device has a thin ring shape, it can be attached to various types of biological microscopes from various manufacturers with a simple attachment.

1 is a conceptual cross-sectional view of a dark field illumination device according to the present invention. The dark field illuminating device perspective view by this invention. A is a conceptual perspective view thereof. B is a conceptual cross-sectional view showing a part of the variable mechanism. The micrograph which compared both the case A when illuminated and image | photographed using the dark field illuminating device by this invention, and B when illuminated and image | photographed with the dark field condenser. The perspective view of the dark field illumination device by this invention which has arrange | positioned the light source in zigzag form. The conceptual diagram of the irradiation range of the dark field illumination device shown in FIG. The perspective view A of the dark field illuminating device by this invention which has arrange | positioned the semiconductor light source in zigzag form, The partial cross section conceptual diagram B. FIG. The perspective view A of the dark field illuminating device which covered the light-shielding tube on the light source by this invention, and the partial cross section conceptual diagram B. FIG. The perspective view A of the dark field illuminating device provided with the reflecting plate near the light source by this invention, and the partial cross section conceptual diagram B. FIG. The perspective view A of the dark field illuminating device using the surface mount type and side irradiation type light emitting diode by this invention, and the partial cross section conceptual diagram B. FIG. The top view of a strip | belt-shaped flexible substrate. The assembly perspective view of a strip | belt-shaped flexible substrate. The perspective view of the ring-shaped frame which mounted | wore with the flexible substrate of FIG. FIG. 13 is a cross-sectional view of a main part of FIG. 12. The top view of a gear-like flexible substrate. The perspective view which mounted the flexible substrate of FIG. 14 to the ring-shaped frame. The principal part sectional drawing of FIG. A is a perspective view of a dark field illumination device of the present invention using a rotating washer, B is a perspective view of a main part of FIG. 17, and C is a cross-sectional view of a main part of B of FIG. A in which the light source is provided on the annular base is a perspective view of the dark field illumination device of the present invention. B is a sectional view of an essential part thereof. The schematic sectional drawing in the case of using the dark field illumination device by this invention as an epi-illumination device. Micrographs of animal brain cells with bright field illumination A, bright field illumination and dark field illumination B and dark field illumination C, respectively. The conceptual diagram of the illumination optical path of the conventional biological microscope. The conceptual diagram of the dark field condenser lens of the conventional biological microscope. Dark field image at low magnification obtained with a conventional dark field condenser lens. The conceptual diagram of the dark field illumination system of a stereomicroscope. A photomicrograph of dark field illumination with a 1 × objective lens of a stereomicroscope. A is a state with a sample. B is the state where the specimen is removed. A biological micrograph showing a flare of dark field illumination of a commercially available 1 × objective lens. The block diagram which compared the prior art and this invention which show the relationship between the objective-lens magnification of a biological microscope and a stereomicroscope, and the dark field illumination range.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Light source, 2, 4 ... Condensing lens, 3 ... Reflecting mirror, AL ... Dark field condenser lens, ML ... Mirror, ST ... Stage, 5 ... Sample, 6 ... Objective lens, 7 ... eyepiece, 8 ... base, 9 ...
Ring frame, 10, 17 ... Light-emitting diode, 11 ... Small piece, 12 ... Support shaft, 13 ... Male screw, 14 ... Female screw, 15 ... Shading cover, 16 ... Reflector, 18 ... Collar Part, 19, 20 ... flexible substrate, 21 ... rotating washer, 22, 23 ... fixing pin, 24 ... stop hole, 25 ... long hole, 26 ... reflective surface,

Claims (1)

The light flux emitted from the plurality of semiconductor light sources arranged on a concentric circle, by irradiating the sample surface placed perpendicular to the optical axis of the observation light, dark used biological microscope that allows dark field observation of the sample A field illumination device having a hollow base, a hollow ring-shaped frame screwed to the base, and a stop hole into which a fixing pin standing on the circumference of the ring-shaped frame is inserted at one end side. wherein the rotary washer to rotate as a fulcrum fixed pin with the rotation of the annular frame, are disposed the rotary washers of the other end side an electrically insulated by a light beam toward the sample surface so as to illuminate respective Te A dark field illumination device for use in a biological microscope, characterized in that the rotating washer rotates with the rotation of the ring-shaped frame to change the diameter of the irradiation region.

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