WO2003063078A1 - Imaging device - Google Patents

Abstract

An imaging device for measuring the number of minute objects that are excited by irradiation light in a plurality of different wavelength areas and produce feeble light having wavelengths respectively different from the wavelength areas of the irradiation light. An imaging device for imaging minute objects to be picked up that emit light, according to irradiation light in at least two wavelength areas, at wavelengths different from the wavelengths of the irradiation light, for conversion into electric signals, the device comprising a stage mounting thereon an object to be picked up including minute objects, a light source means for irradiating the object-mounting surface of the stage, a light switching means for switching irradiation light beamed from the light source means to rays of light in a plurality of different wavelength areas, a lens means for condensing light from the object irradiated with rays of light in the different wavelength areas, a photoelectric conversion means for converting a two-dimensional image light condensed by the lens means into an electric signal, and at least two filter means switched according to the switching of rays of light by the light switching means, the filter means being disposed between the lens means and the irradiation light emitting position of the light source means on the light path.

Description

 Technical field

 According to the present invention, when light of a plurality of different wavelength regions is irradiated, the light is illuminated by the irradiation light.

The present invention relates to an image pickup apparatus for picking up an image of a minute individual that is excited and generates light having a wavelength different from the wavelength region of the irradiation light (hereinafter referred to as “fluorescence”) and converts the image into an electric signal.

 book

 Background art

 When bacteria are given special reagents and irradiated with light in a plurality of different specific wavelength regions, the pateria are known to produce fluorescence in a wavelength region different from the irradiated light depending on their life and death. I have. For this reason, a specimen containing bacteria to which such a special reagent has been given is loaded on a stage, the fluorescence of the specimen is imaged, and the obtained image data is subjected to image processing to thereby determine the number of battery cells in the specimen. It is possible to count.

 The brightness of the fluorescence of the battery is usually weak, but increases with the amount of light applied. Therefore, in an imaging device for imaging the fluorescence of bacteria, in order to receive fluorescence of illuminance of a predetermined amount or more from the bacteria in the sample, the stage on which the sample is loaded is irradiated uniformly and with an illuminance of a predetermined amount or more. There is a need.

In such an imaging apparatus, the photoelectric conversion means (means for converting an optical image into an electric signal; hereinafter, appropriately referred to as an “image sensor”) such as a CCD camera must be directed in a direction to observe the fluorescence of bacteria. Therefore, when observing the image sensor from above the sample loading stage, light cannot be illuminated from below the stage. For this reason, light is illuminated obliquely from above the stage, but in order to irradiate light on the stage with uniform illuminance and at a predetermined illuminance, it is necessary to irradiate light from multiple positions. . For this reason, in the imaging device of the prior art, in order to irradiate the stage with high illuminance light, the irradiation light from the light source is used. Light was shining on the stage.

 By the way, since a light source for irradiating light on a stage in such an imaging apparatus needs light in a specific wavelength region, it is possible to use a light emitting diode suitable for this. In recent years, light-emitting diodes that emit high-brightness light in a wide wavelength range have been developed.If this is used as a light source, it must be combined with a color filter that emphasizes light in unnecessary wavelength ranges. is there.

 On the other hand, in an image reading device such as an image scanner for reading an image of a document document or the like, a three-color switchable color separation is performed in an optical path between an imaging lens and an image sensor. A color filter is arranged. The color image reader reads the image data, for example, for each of the three primary colors in the document document by switching between the three color filters. Japanese Patent Application Laid-Open No. 2000-322214 discloses an example of such a color image reading apparatus. In the device disclosed herein, the thickness of a color filter in the form of a photoelectric conversion chip in a color image sensor is changed to adjust the optical path length for each color. However, if the wavelength range of the light source is limited, such a method can be used.However, when light in a plurality of wavelength ranges is imaged by the same imaging means, such as fluorescence from a battery, Such a method of adjusting the optical path length for each color cannot be adopted because the structure becomes very complicated and the apparatus becomes large. Also, a method of reducing chromatic aberration by designing a combination of a plurality of lenses having different refractive indexes complicates the structure and increases the cost. The same applies to the configuration in which the switching filter is inserted into the lens unit (lens means).

 By the way, in an imaging apparatus for imaging weak fluorescence from bacteria in a specimen, a magnifying optical system has to be employed in order to precisely image a subject having a weak light amount. Therefore, in such an image pickup apparatus, the depth of focus is extremely small, and the adjustment of the focal position for each light receiving wavelength range is indispensable. In addition, when imaging is performed for each fluorescence having a plurality of wavelength regions, if there is any defocus in each of the imaging data, the outline of the pateria cannot be grasped on the basis of the imaging data, and the number of individuals of the nocteria can be reduced. Accurate counting cannot be performed.

Furthermore, in such an imaging device, the size of the individual to be imaged is extremely small. For this reason, the stage must be divided into a plurality of imaging unit areas for imaging (several micrometers). For this reason, an image is taken while the stage is moved in the X and Y directions. In order to image the outline of the individual precisely and clearly, the distance between the imaging surface and the condenser lens is fixed (for example, It is necessary to maintain the focal length of the lens within the range of (number + microphone). Further, when a plurality of types of collection kits for mounting a sample are prepared, the distance between the imaging surface and the condenser lens must be kept constant according to the difference in the shape and the like of the collection kit. Disclosure of the invention

 An object of the present invention is to provide an imaging apparatus capable of imaging weak fluorescence from a subject having a plurality of different wavelength regions having various problems as described above by using a mechanism as simple as possible.

 The present invention also provides such an image pickup apparatus, in which an almost uniform irradiation of the entire imaging region is realized by a small-sized stage and a fixing means having a simple structure without mounting an automatic focus adjustment device, It is an object of the present invention to provide an imaging device that can keep a distance between an imaging surface and a collection kit within a certain depth of focus range corresponding to a plurality of types of collection kits.

 The present invention further irradiates a uniform amount of light of a predetermined amount or more on the upper surface of the stage on which the sample is mounted, and easily adjusts the position of the light source in relation to the spot center of the light source and the optical axis of the imaging camera. It is an object of the present invention to provide an imaging device capable of performing such operations.

For this reason, the present invention is an imaging apparatus that captures an image of a minute subject that emits light in a wavelength region different from this wavelength region in accordance with irradiation light of at least two wavelength regions and converts the image into an electric signal. A stage for loading a subject including an individual, light source means for illuminating a surface of the stage on which the subject is loaded, light switching means for switching irradiation light emitted by the light source means to light rays of a plurality of different wavelength regions, A lens means for condensing light from the subject irradiated by the plurality of light beams having different wavelength ranges, and an image sensor for converting the two-dimensional image light condensed by the lens means into an electric signal Is switched according to the switching of the light beam by the light switching unit. An imaging apparatus, comprising: two or more filter units, wherein the filter unit is disposed in an optical path between the lens unit and a position where the irradiation light of the light source unit is emitted. .

 As a result, the present invention provides an imaging device for imaging light in a plurality of different wavelength regions from a subject including an individual, in which a difference in optical path length caused by a difference in the wavelength region can be adjusted with a simple mechanism. As a result, the outline of a minute-sized individual was precisely achieved. Furthermore, by arranging two or more filter means in the optical path between the lens means and the position where the light emitted from the light source means emits light, even if the lens means is replaced with one having a different magnification, the optical path length can be easily adjusted. This made it possible to readjust.

 The present invention also provides a stage on which a collection kit including an individual to be imaged is loaded, and a fixing means provided on the stage so as to be openable and closable, and fixing the collection kit and providing an imaging opening. Light emitting means; light converging means for converging light from the light emitting means and projecting it as a light beam onto the stage; light source means for irradiating the stage with light; and a direction substantially perpendicular to the stage. A lens means for condensing light from the individual, and a photoelectric conversion means for converting the light condensed by the lens means to an electric signal. An imaging device is provided, wherein the imaging opening is opened such that the light beam emitted from the light converging means is applied to the entire region to be read on the stage. It is intended.

Accordingly, in the imaging apparatus according to the present invention, substantially uniform light beams can be radiated to the entire imaging area by the small-sized stage and the fixing means having a simple structure without mounting the automatic focus adjustment apparatus. This enabled the distance between the imaging surface and the sampling kit to be within a certain depth of focus range, corresponding to each type of sampling kit. The present invention further includes a stage for loading a subject including an individual to be imaged, light source means for irradiating a light beam to a stage surface of the stage, and condensing light from the subject irradiated by the light source means. A lens unit that converts the two-dimensional image light condensed by the lens unit into an electric signal, and the light source unit irradiates the stage surface with light obliquely from above. Rays Means, holder means for supporting the plurality of light means so that light emitted from the plurality of light means irradiates substantially the same position on the stage surface, holder means and the lens means And a fixing means for integrally fixing and, and an image pickup apparatus characterized by having: Here, the light beam means includes a first light beam means having a light beam means for emitting a light ray in a first wavelength region, a second light beam means having a light beam means for emitting a light beam in a second wavelength region, Wherein each of the first light beam means and the second light beam means further comprises a plurality of light beam means for irradiating the stage surface with light beams from a plurality of directions symmetrical to the lens means. It is composed.

 As a result, the present invention irradiates the upper surface of the stage on which the sample is mounted with a uniform amount of light of a predetermined amount or more, and easily adjusts the position of the light source in relation to the center of the light spot and the optical axis of the imaging camera. Thus, an image pickup device capable of performing the above was realized. Brief description of the drawings

 FIG. 1 is a side view of an imaging device of the present invention and a data processing device connected to the imaging device.

 FIG. 2 is an overall perspective view of the imaging apparatus.

 FIG. 3 is a plan view mainly showing the vicinity of a light source when the imaging device of FIG. 1 is viewed from above. FIG. 4 is an enlarged side view showing a part where a lens unit (lens means) and a light source (light, di means) are attached to a flange (holder means).

 FIG. 5 shows a sectional view of the internal structure of the rod light source.

 FIG. 6 is a plan view of the stage of the imaging apparatus when observed from above. FIG. 7 shows (a) a side view and (b) a cross-sectional view in which an emblem kit is mounted on a stage of the imaging apparatus.

 FIG. 8 shows (a) a side view and (b) a cross-sectional view in which a film kit is mounted on a stage of the imaging apparatus.

FIG. 9 is a diagram for explaining details of the imaging opening of the kit holder. FIG. 10 is a control block diagram of the imaging apparatus. FIG. 11 shows a perspective view of the filter moving means.

 FIG. 12 shows a configuration diagram of a subject, a filter unit, and a lens unit. FIG. 13 shows a flowchart of the imaging operation of the present apparatus.

 FIG. 14 is a diagram showing terminals of an imaging means (image sensor).

 FIG. 15 is a flowchart showing background level removal.

 Figure 16 is a graph showing the exposure time at the ideal subject (bacteria image) level.

 FIG. 17 is a diagram showing the appearance of “bright spots”.

 FIG. 18 is a diagram showing the line of the “fungus + background” level and the background level of each frame.

 FIG. 19 is a diagram showing a usable range of a pixel frame.

 FIG. 20 is a diagram showing that background outputs overlap and white noise and the like specific to semiconductors are superimposed.

 FIG. 21 is a diagram showing that Β 黒 is the black level in the ith frame, and that the white noise person Nw is always superimposed regardless of the exposure.

 FIG. 22 is a diagram showing that white noise soil Nw is superimposed on the video level of a certain bacterium.

 FIG. 23 is a diagram showing a list of various forms of the adding means.

 FIG. 24 shows the wavelength relationship between irradiation light and exposure (including filter characteristics) to the photoelectric conversion element. BEST MODE FOR CARRYING OUT THE INVENTION

 Here, before describing the details of the imaging apparatus of the present invention, its technical background will be described. (1) Technical background of the imaging device

This imaging device is an inspection system equipped with an optical system and a photoelectric conversion element such as a CCD camera, which is used for acquiring images containing bacteria (fungi) and examining the numbers of living bacteria and dead bacteria. Device. In addition, there are several types of photoelectric conversion elements (photoelectric conversion means) other than CCDs, such as a MOS type, but since the present invention is basically applicable, the type is limited to CCDs. Do not mean. Normally, a general camera captures the reflected light from the illuminated subject and obtains image data for the image. In this system, however, a special chemical solution (reagent) is included in the specimen, which is the measurement object in the subject. In this method, the photoreaction depends on the state of the living or dying bacteria to be detected. This photoreaction is photoexcitation caused by light of a specific wavelength, and the energy of the fluorescence generated by bacteria is very small compared to the light energy to be irradiated. I can't do it. If the strong irradiation light enters the photoelectric conversion element as it is, it will be saturated. Therefore, it is necessary to cut between the irradiation light and the fluorescence of bacteria excited by the irradiation light. Further, after the light is cut, the exposure time of the photoelectric conversion element is set to be considerably longer than usual in order to acquire weak fluorescence and convert the image data into image data, so that the charge is sufficiently accumulated in each photodiode. Required. In addition, it is necessary to remove background noise and white noise generated at this time.

 By the way, the charge storage time in the photodiode constituting each pixel is on the order of several tenths of a second in a normal standard, whereas in the case of the present example, it may be several seconds. In such a usage form of the photoelectric conversion element, a phenomenon of generation of a bright spot occurs due to imperfect formation of a semiconductor.

 A luminescent spot is a pixel whose charge is not normally accumulated in a photodiode in response to light input, or which has an abnormally high voltage output due to an abnormal process of charge accumulation and transfer. Means When a bright spot occurs, the bright spot is treated as if it were fluorescent light generated by a pattern.If the size of the detection target is smaller than a few pixels, the relevant bright spot is mistaken for the detection target. Weave.

 If you proceed to the process of detecting the number of individuals while including these bright spots, the wrong count will be counted. In order to avoid this, we propose to make the bacteria image clear before image processing.

 (2) Explanation of bacteria that emit fluorescence of a specific wavelength

The background (where the individual is not present in the specimen) is a resin-containing specimen or a film with an adhesive layer to adhere bacteria. Both are colored as dark as possible in order to obtain the fluorescence of the batteria efficiently (with good contrast). It is dark gray rather than completely black, When emitted, it produces reflected light.

 In this device, the inspection is not performed with the reflected light of the irradiated light, but a special chemical solution is contained in the pat- ter, and ultraviolet light or green light is irradiated in this state. In response to the ultraviolet light or green light, bacteria containing the chemical solution generate fluorescence. Specifically, as shown in (a) and (b) in Fig. 24, when ultraviolet light with a dominant wavelength of 370 nm (N SHU 590) is irradiated, bacteria emit fluorescence with a dominant wavelength of 461 nm. . When green light with a dominant wavelength of 525 nm (NSPG500S) is irradiated, fluorescence with a dominant wavelength of 617 nm is generated.

 FIG. 24 (c) shows the spectral characteristics of the filter. Fluorescent light is weak to the illuminating light, and because the wavelengths are close to each other, the wavelength of the illuminating light must be adjusted in the light path to receive light.

 For this reason, UV light is attenuated from 360 nm and is irradiated with a cut filter (U360), which has the characteristic of attenuating the transmittance to 0% at the point of about 400 nm. Set the transmittance to 0% at the point. This puts the little power that the light source LED NS HU590 emits at the 400 nm point almost to power. Then, on the light receiving side, a filter called L42 that transmits a wavelength longer than 420 nm is inserted. Then, fluorescence with a dominant wavelength of 461 nm can be acquired properly. This fluorescence occurs with live and dead bacteria.

 Similarly, the green side uses a filter. For green light, a bandpass filter BP 535 that transmits only from 450 nm to 550 nm is attached to the green light source (dominant wavelength 536 nm). The light source LED NS PG 500 S emits light with a dominant wavelength of 525 nm from 450 nm to 650 nm, but its tail is emphasized by the bandpass filter described above. On the light receiving side, insert a filter called O58 that transmits 580 nm or more. Fluorescence with a dominant wavelength of 617 nm can be obtained. This fluorescence is generated only for dead bacteria. Since the intensity of this fluorescent light is weak, it requires an exposure time of about 2 seconds, during which the bright spots described above are generated. Therefore, it is preferable to proceed to the counting of the number of individuals after performing the processing for suppressing the generation of the bright spot.

Hereinafter, an embodiment of an imaging device according to the present invention will be described in detail with reference to the drawings. In this device, instead of acquiring a color image at the same time, the color filter is turned off. This is a device that acquires monotone images for two wavelengths. The subject is irradiated with irradiation light of two different wavelengths (ultraviolet light and green light) from two pairs of light sources. In other words, all pixels have the same wavelength at the acquisition timing of certain image data, and the difference in optical path length for each wavelength is constant unless the lens system is changed. It is optional to do so. However, in future developments, as the types of imaging objects increase, if the lenses are configured to be interchangeable for the purpose of optimizing the magnification, the amount of optical path length correction will change, and adjustment means will have to be replaced. Needless to say.

 From this relationship, the object, the switching filter, the lens unit, and the image sensor are configured in this order, and the switching filter has a configuration in which the thickness of each of two different wavelength filters is changed.

 FIG. 1 is a side view of an imaging apparatus 1 for counting the number of individuals of a subject according to the present invention, and an entire data processing apparatus 100 connected to the imaging apparatus 1.

 FIG. 2 is an overall perspective view of the imaging device 1. As shown in FIGS. 1 and 2, the imaging apparatus 1 performs command control, and is connected to a data processing apparatus 100 that determines the number of individuals such as bacteria from an image via an interface cape 101. I have. The imaging device 1 includes, as main components, a stage 2 on which a subject to be inspected is placed, a front cover 5 which is opened and closed for operations such as placing a subject (sample) on the stage 2, and the like. A light source unit 4 for irradiating the subject with light, and a lens unit 3 for receiving two-dimensional image light from the subject, which is illuminated by the light source unit 4 and emits fluorescence by a reagent, and converts the two-dimensional image light into image data of electric signals. A filter moving unit 23 that supports a plurality of filters and moves to switch the filters according to the switching of light emission; and a filter moving unit 23 that moves the position from the operability when the sample is placed on the stage 2. Stage moving part 9 and power.

Stage 2 consists of an emblem kit 40 (first collection kit, shown in Fig. 7) or a film formed between the upper surface of the upper tape 20 Install kit 41 (second collection kit, shown in Figure 8). Kit Honoreder 21 is fixed with lock lever 22. The upper table 20 is fixed on the X table 25, and the X table 25 is fixed on the Y table 26. It is. The upper table 20 on the X table 25 is supported at four points by elastic plates (panels) 35 at four places. The positioning jig is temporarily fixed, and the upper table 20 that is energized by the panel is this jig. Is brought into contact with it, and it is made to be level. The upper table 20 is screwed by the lock plate 36 while positioning. After that, different jigs are attached to the lens unit 3, and the final alignment of the stage 2 (upper table 20) and the lens unit 3 is performed.

 The stage moving section 9 is provided with an X1 sleeve 31 and an X2 sleeve 32 that are slidably fitted to the X1 shaft 27 and the X2 shaft 28 at the lower part of the X table 25, respectively. The lower part of the Y table 26 is provided with a Y1 sleeve 33 and a Y2 sleeve 34 that are slidably fitted to the Y1 shaft 29 and the Y2 shaft 30, respectively. Each is driven by the belt by the X drive unit 8 and the Y drive unit 7, respectively. The power is turned on to the device by the power switch 10, and the initial operation starts. In the initial operation, the stage 2 is first moved to the reference position. This reference position is a position where the subject is imaged, and is set directly below the lens unit 3.

 Stage 2 has X—HP sensor 1 that indicates the reference position in the X direction along the X1 shaft 27, X2 shaft 28, Y1 shaft 29, and Y2 shaft 30 shaft directions. Move to the position where both 1 and Y-HP sensor 1 and 2 indicate the reference position in the Y direction. When the initial operation is completed, a standby state is entered. When the front cover 5 is opened, a signal indicating that the operator has opened the front cover 5 is issued from a cover sensor (not shown). According to this signal (cover open signal), the stage 2 moves to the front position where the front cover 5 is opened. The open state of the front cover 5 is maintained by the coil spring 5a.

When the subject (sample) of the emblem kit 40 or the film kit 41 is mounted on the stage 2 and the front cover 5 is closed by the operator, the signal of the cover sensor is transmitted to the data connected via a predetermined interface. It is sent to the processing unit 100 and displays on the display that the preparation is completed. The operator instructs the data processing device 100 to start measurement. The instructed data processing device 100 sends a measurement start command to the imaging device 1. The control unit of the imaging device 1 controls the Y drive unit 7. To move the stage 2 to the initial shooting position, which is the home position for shooting. In this example, the data processing device 100 is a personal computer.

 The photographing of the object to be photographed in this apparatus is repeatedly performed while the position of the stage 2 is gradually changed. The difference between measuring the emblem kit 40 and measuring the film kit 41 is different.However, if the external shape of the specimen, which is a sample, is extremely small with a size of several microns, for example, the entire area to be photographed is the emblem kit 40. In this case, the area is 12 mm * 9 mm, and the area that can be acquired in one shot is 1.2 mm * 0.9 mm, so 100 shooting frames are required. In the shooting process, shooting is performed 10 times while moving in the Y direction, and is repeated in the X direction.

— Each time one frame is captured, the image data is transmitted to the data processing device 100. After completing the acquisition of the image data, the data processing device 100 requests the imaging device to acquire the next frame, whereby the imaging device performs position movement and re-shooting. In this way, necessary frames are repeatedly photographed. After all necessary frames have been captured, Stage 2 returns to the home position described above. A detailed description of this imaging operation will be described later.

 Fig. 3 is a plan view of the light beam unit 4 as viewed from above and below the device. Fig. 4 is an enlarged view of a portion where the lens unit 3 and the light source 6 are attached to the flange 51 (holder means). Is shown.

 The light source, part 4 includes a pair of a mouth light source 6a and a mouth light source 6b as a first irradiation means, and a pair of a rod light source 6c and a mouth light source 6d as a second irradiation means. It consists of books. Each of the aperture light sources 6 has a substrate 50a, 50b, 50c, 50d at an end, and a light emitting diode is installed on the substrate facing inward of the aperture. As a first irradiation means, the light source 6a and the rod light source 6b (NSHU590) emit light of the same wavelength (ultraviolet light having a wavelength of 370 nm) and work as a pair. A second light source is a mouth light source. And the rod light source d (NSPG500 S) emit light of the same wavelength (green light with a wavelength of 525 nm) and work as a pair. ,

As already mentioned, the reason that the two rod light source pairs (6a and 6b) are placed at the target position is to complement each other to make the illuminated surface almost uniform. The light source pair (6a and 6b) and the rod light source pair (6c and 6d) cannot be turned on at the same time. And need not be orthogonal.

 With this configuration, the six pairs of rod light sources irradiate the irradiation stage having the imaging means arranged above, so as to have the same illuminance at the same wavelength and diagonally from two force points symmetrical to the stage on the side of the imaging means. At the same time, the projection area matched the irradiation stage so that there was no uneven illuminance.

 Then, the stage 2 is located below the light emitting end opposite to the end where the light from the light source unit 4 is mounted on the substrate. A lens unit 3 of lens means is provided above the light emitting end, and a filter unit 55 that advances and retreats in parallel to the surface of the stage 2 is located between the lens unit 3 and the light emitting end. The filter unit 55 is equipped with an L42 finalizer 55b that cuts the wavelength band below 420 nm and an O58 filter 55a that cuts the wavelength band below 580 nm. ing. With this filter unit 55, the lens unit 3 can obtain clear fluorescence in which the overlapping wavelength bands are cut.

 The sample to be tested for a battery is a sample that is fixed on a sample device called an emblem kit that can absorb water after the object has been stirred in the solution and then absorbs the test reagent. In some cases, it was directly collected on a film having Further, it may be possible to consider a fixing device having a different shape. As described above, since the enlarged image is formed on the photoelectric conversion element 96 (Fig. 12) using the magnifying optical system and photographed, the distance to the subject is extremely strict due to the small depth of focus. Is done.

 For this reason, Stage 2 has four adjustment points to adjust not only the height but also the level. An attachment portion (not shown) of the lens unit 3 to the frame has an elongated hole so that an approximate adjustment can be performed so that the adjustment margin can be adjusted at these adjustment points. These adjustments determine the position of the stage and the position of the image sensor.

The rod light sources 6a, 6b, 6c, and 6d, which are four rod-shaped light sources, are configured to be attached to one mounting flange 51 (holder means). The mounting flange 51 has an X-shape when viewed from above, and includes a ring portion 57 for mounting the lens unit 3 and four arms 5 6a radially protruding from the ring portion 57. It consists of 56b, 56c and 56d.

 The arms 56a, 56b, 56c, 56d are respectively provided with the mountings 62a, 2b, 62c, provided on the rod light sources 6a, 6b, 6c, 6d. And 6 2 d are provided with a mouth mounting part so that they can be adjusted and fixed by screwing.

 Each of the four mounting portions of the flange 51 is formed such that when the mouth light source 6 is mounted, the irradiation light of the mouth light source 6 has an angle positioned on the optical axis of the lens means. In this example, four rod light sources 6 a, 6 b, 6 c, and 6 d are used, but the number of the rod light sources in the present imaging device is not necessarily limited to the four rod light sources exemplified above. The number is not limited to six or eight, for example. Here, when the number of rod light sources is 6, every other 3 of the 6 light sources have ultraviolet light at the position corresponding to the vertex position of the equilateral triangle, and the other 3 light sources have Green light is provided at the position corresponding to the vertex position of the equilateral triangle.

 A pair of open light sources that emit irradiation light of the same wavelength irradiates the lens unit 3 symmetrically to the lens unit 3 on the stage 2 and obliquely from the opposite position to make the irradiation surface almost uniform in brightness. ing. Each port light source 6 is fixed to one flange 5 1 so that the power of the two rod light sources can be evenly distributed over the entire photographing area on the stage 2 where the subject is photographed. The steps 52 a, 52 b, 52 c, and 52 d formed on the outer wall of each rod are engaged with the flange 51 to perform positioning.

 Furthermore, not only the position, but also the LED, which is the light source means, has large variations in product accuracy, and when used at the same operating voltage, there are illuminances that are about twice as different.Therefore, the control voltage is sent by sending data from the CPU to the DZA. The illuminance is adjusted by adjusting the current for each line.

To attach the mouth light source 6 and flange 51, engage the mouth light source 6, the step 52 of flange 6, and the mounting part of the flange 51, adjust the position, and then use the bracket 6 2 to attach the flange. 5 Screw it to the mounting part of 1. This step is machined so that the tip of the rod light source 6 is positioned at a desired position with almost no design adjustment. If it is necessary to further adjust the position of the light source after screwing, the screws are loosened to facilitate adjustment. Thus, in the light source means of the present invention, the convergence position of the light is located on the optical axis of the lens means. It is configured to be adjustable to be placed. In this light source means, a unit assembling step of the light source 6 and the flange 51 is performed in a light source means assembling step before an assembling step to the lens means. At this stage, the adjustment was almost completed, and by attaching the light source unit 4 to the lens means, the adjustment of the positions of the lens means and the light source means was completed, and the adjustment process was simplified.

 FIG. 5 is a new view showing the structure of the mouth light source 6 using the mouth light source 6a as an example. The colors, lenses, etc. of the light-emitting diodes of the mouth light source 6 differ between the 370 nm light source and the 525 nm light source. The basic structure is the same. It can be broadly divided into a rod light source 60a and a rod light source 61a having a smaller diameter. At one end of the light source 60a, a light emitting diode 64a is fixed to the substrate by soldering via a diode spacer 63a to the substrate 50a.

 Light emitting diode 6 A lens fixed with a ring 7 2a ahead of the light emitting direction of 4 a 6

There is 5 a. The mouth light source 61a is inserted into the other end of the mouth light source 60a. At the end of the rod light source 6 1a penetrating the rod 60, a ring 7 4a and spacer are attached.

There is a lens 66 a fixed at 67 a and a band cut filter 73 a. The band cut filter 73a has a function of cutting an unnecessary foot portion in each light emission distribution, and the U360 filter 73a is provided in the rod light sources 6a and 6b, and similarly in the 5 35 5 is provided in the rod light sources 6 c and 6 d to sharpen the light emission distribution.

 At the other end of the rod 61a, there are a lens 68a and a lens 69a fixed with a ring 70a and a spacer 71a. Further, a mounting portion 62 a for mounting to the flange 51 is provided. Then, a step 52 a formed by a difference in diameter between the rod 60 a and the rod 61 a is engaged with the flange 51. The mounting of the rod 61 is accurately positioned with respect to the mounting position of the rod of the flange 51 by the step 52a.

In addition, as described above, the rod light source is not fixed to four rods, and is used not only for the light source used by switching to two wavelengths but also for spot irradiation as a light beam without switching. The purpose is to match the center to the optical axis of the lens unit (lens means) 3 so that the fluorescence of bacteria can be imaged. The present invention is not limited to such a device.

 FIG. 6 is a diagram in which the stage 2 of the imaging apparatus is observed from above. The stage 2 is irradiated with light from the light source unit 4. The light source unit 4 is composed of four rod light sources 6a, 6b, 6c and 6d. Each of the mouth light sources 6 has a substrate 50a, 50b, 50c, and 5Od at an end.

 The stage 2 is provided with fixing means 21 for holding the sampling kit, and an imaging opening W is provided at the center of the fixing means 21. The stage 2 moves in the X and Y directions in the horizontal direction. When the stage 2 is located at the initial reading setting position (center position), the center of the imaging aperture W is imaged through the lens unit 3. The center (so-called optical axis) is made to coincide. Further, the fixing means 21 corresponds to the lens unit (lens means) 3 corresponding to substantially the same position on the stage surface of the stage 2 illuminated by the plurality of light sources (light means) 6. It is configured to be movably supported.

 To move from the center stop position, the stage 2 moves right and left and up and down from the center by 8.5 mm, a half of 17 mm in both X and Y directions. For this reason, the imaging opening W is required to be 17 mm wider than the imaging region of 17 mm.

 The size of the opening W is such that the illumination light from the light source 6 enters obliquely, and it is necessary to retract the edge of the window so as not to be blocked by the thickness of the fixing means 21 having the window. Therefore, the size of the opening W must be further enlarged.

 Here, if the imaging opening W is circular and has the same size as the area for reading, the light (light ray) from each rod light source 6 will be blocked. For this reason, the diameter of the imaging aperture W must be larger than the diameter of the 34 mm circle so as to be larger than the beam diameter so that the irradiation light from the rod light source 6 is not blocked.

 In this case, the size of the opening W becomes considerably large. For example, the lock portion 21a of the intermediate plate 42 used when the film kit is used is further lowered below the drawing shown in FIG. The necessity increases the size of the equipment, affecting the surrounding design.

However, as shown in Fig. 6, when the opening W is opened in a square area, the bulging angles Wa, Wb, Wc, and Wd allow a wide range of light irradiation, and the side The overhang is small and the device can be made more compact.

 The kit holder 21 for fixing the collection kit on the stage 2 is equipped with an elastic body. By directly fixing the collection kit (emblem kit 40 or film kit 41) itself, the stage 2 Can be eliminated while moving in the XY direction.

 The fixing elastic body (set leaf spring) is an elastic plate (panel) 25 2 and is attached to the kit holder 21 and the contact means (pin) attached to the kit holder 21 to the slider bull. In conjunction with, fix by pressing the sampling kit. If an attempt is made to provide a projection on the fixing elastic body that comes into contact with the contact pin, processing failures such as cracks may occur. In order to avoid this, no protrusion is provided on the fixing elastic body, and a protrusion is provided on the contact pin side in this embodiment. Further, operability can be improved by attaching a handle to the film holding plate.

 To prevent forgetting to close the kit holder 21, when moving from the front at the start of measurement, the kit holder 21 automatically closes with the movement by engaging with the fixing plate holding means above the entrance near the case. I have.

 The stage 2 on which a sample is placed is different only in the type of collection kit to be set in the kit holder 21. The basic structure of both collection kits is the same. However, in the case of the film kit 41, the hole for accommodating the loading of the emblem kit 40 is closed, and the intermediate plate 42 (mounting means) having a height adjusting function is loaded.

 FIG. 5 shows a state in which the en-prem kit 40 is mounted on the stage 2 of the imaging apparatus 1. In the emblem kit 40, an injection f | ", which is not shown in the figure, is connected to the measurement unit and extends downward. The object to be inspected is finely crushed and immersed in water to suck up the solution or directly remove the liquid in food. It is possible to observe the presence or absence of bacteria by impregnating the measuring part with the sucked liquid Even if the solution is spilled from the injection needle onto the device, there is no effect on the device As described above, the cover 20 c is provided on the lower surface of the stage 2.

FIG. 8 shows a state where the film kit 41 is mounted on the stage 2 of the imaging apparatus 1. Film kit 4 1 has an adhesive surface and a protective film is deposited during storage. However, it should be removed from the specimen when used.

 Since the emblem kit 40 has a measuring portion above the flange portion 40a, the emblem kit 40 is positioned higher than the flange portion 40a. On the other hand, the film kit 41 is in the form of a film and does not have such a height (thickness). Furthermore, the film may bend depending on how it is held down. On the other hand, since the lens unit 3 is not only a magnifying optical system but also the contour of the individual is important for the measurement of the number of subjects, the depth of focus of photography is extremely narrowed. Moreover, since the size of the bacteria to be imaged is small and is of the order of xm, there is only about one pixel (one pixel is 1.6 μιη). Therefore, the height of the imaging surface needs to be set at the same level position in the two types of sampling kits.

 In order to adjust the level position, when the film kit 41 is mounted, the intermediate plate 42 is used also as a support for the film. In other words, since the stage 2 has a hole 20a for inserting the emblem kit 40, if the film kit 41 is loaded as it is, the center will sink and the desired focal position will be set. Is no longer obtained. The light emitted from each of the rod light sources 6a, 6b, 6c, and 6d does not contain many infrared components, but is not completely absent. If loaded, subsidence in the center would be accelerated.

 FIG. 9 is for explaining the details of the imaging opening W of the kit holder 21. FIG. 9 (a) shows a top view of the kit hono-redder 21 and FIG. 9 (b) shows a cross-sectional view of the kit hono-leder 21.

 As shown in FIG. 9A, the kit holder 21 has an opening (imaging opening) W O. The shape of the imaging opening W O is substantially square except for the cut C formed in the side. Symbols L1, L2, L3, and L4 in the figure indicate irradiation spots (light rays) from each light source.

In the present embodiment shown in FIG. 9, since the stage 2 moves with the imaging position fixed, the area W1 indicated by the broken line when the state shown in FIG. W2 moves to capture an image. Then, taking the X direction as an example, at least, if there is a window in the area W1, the light beam will not be blocked, but it will move by M, so The imaging aperture must also be increased by M. Therefore, the actual imaging opening has a size of WO.

 As described above, since the imaging aperture becomes large, in order to pursue a little more compactness, if the apex angle of the rectangle is located in the direction in which the light beam is projected, the position of the side that is not related to the projection Should be cut out like C.

 The kit holder 21 is fixed between the intermediate plate 42 and the sampling kit holding pin 251, which is urged by the elastic plate (panel) 25, holding the film kit 41 at point P. By bringing the sampling kit holding pin 2 51 closer to the irradiation position S 1, the size can be reduced, and the stability of the subject in the area W 3 to be read increases.

 To explain further when moving in the X direction, in order to set the rightmost column in the entire reading area W2 to be read, the imaging opening W0 is moved to the left and the wall A1 is moved to A2. Move to the line position of. Therefore, the light spots L l and L 3 are not obstructed. Similarly, in order to set the leftmost column in the entire reading area W2 to be read, the imaging opening W0 is moved to the right, and the wall surface B1 is moved to the line position of B2. I do. Therefore, the light spots L 2 and L 4 are not obstructed.

 Therefore, when the stage does not move, the minimum imaging opening W1 is, in other words, the area where the light beam is not blocked even when the stage (imaging opening) moves in any direction at the time of maximum movement. I can say.

 Furthermore, since the light source unit 4 of the present apparatus emits light with the light source arranged diagonally, the imaging opening W of the kit holder 21 is used to allow the object to be read to be seen in the direction of the imaging means. The aperture was formed in a square shape with the direction in which the light source was arranged at the apex. By making the imaging opening W rectangular, irradiation with unobstructed light rays can be achieved with a minimum area while securing the surrounding flesh.

By the way, as shown in FIG. 9 (b), the kit holder 21 has a considerable thickness. This is to ensure the rigidity required to tighten the depth of focus and the rest position. Accordingly, since each ray entering obliquely is collected and irradiated to the central spot S1 within an unobstructed range, at least a window in a region W1 indicated by a wavy line is required. This is when stage 2 does not move, This is because the small reading area W3 located substantially at the center of the spot S1 is repeatedly read while moving in the X and Y directions in the entire reading area W2. As described above, in FIG. 9, the entire reading area W 2 is drawn as a square for easy understanding, but the area including the individual to be detected is not necessarily a square. To the extent that the presence of the individual is guaranteed. Further, in the example of FIG. 9, the film kit 41 is used as an example, but an intermediate plate 42 is provided below the film kit 41. (See Fig. 8)

 FIG. 10 shows a control block diagram of the present apparatus. The operator instructs the data processor 100 (see FIG. 1) connected to the interface 80 to start measurement. The instructed data processing device 100 sends a measurement start command to the imaging device 1. The control means 81 that has received the measurement start command via the interface 80 transmits measurement instruction data to the exposure control means 82. Control means 81, adding means for cumulatively adding image data received a plurality of times with the same or different exposure times, counting means for counting the number of individuals (for example, the number of bacteria) based on the image data, And a clock means for controlling the exposure time, a pixel value hierarchy dividing means, a background position specifying means, a background position calculating means, and a filter moving means.

 The counting means need not be in this imaging device, but rather it may be more convenient, if any, in the data processing device 100 to be partly or entirely present as a program. Counting the number of individuals requires a high-speed real number operation when the contour extraction method is used to identify the white area existing in the black background, and this requires an external device such as a personal computer. This is because it is preferable to use a data processing device. It is also convenient in terms of upgrading this algorithm. In this embodiment, the added image data is sent to the data processing device 100 via the interface 80, and the number of individuals (for example, the number of bacteria) of the subject is determined based on the image data in the data processing device. Is counted by the counting means. Fig. 23 shows a list of modes in which such individual number adding means and counting means are mounted.

Further, in the imaging apparatus 1 of the present invention, the control means 81 controls the exposure instruction data instructed to each rod light source 6, the irradiation instruction data transmitted to the irradiation means 83, and the current If it is necessary to move the filter unit 55 based on the current position of the filter unit 55, a move instruction is issued to the filter moving unit 23.

 The fluorescence of the specimen on the stage 2 that emits light by the irradiation light is collected by the lens unit (lens means) 3 and transmitted to the photoelectric conversion means 84. The photoelectric conversion means 84 transmits the received two-dimensional image signal to the A / D converter 85 to convert it into binary image data, and the AZD converter 85 converts the digitally converted data into measurement data. To the frame memory 86. When the irradiation is completed, the frame memory 86 transmits the stored data to the control means 81.

 Here, the mechanism for adjusting the optical path length in the present invention will be described.

 Stage 2 is located below the light-emitting end of the light source unit 4 opposite to the end where the light from the light-emitting unit 4 is transmitted. A lens unit 3 is provided above the light emitting end, and a filter unit 55 that advances and retreats in parallel with the surface of the stage 2 is located between the lens unit 3 and the light emitting end. In the filter unit 55, the light in the wavelength band below 420 nm is turned on.The L42 filter 55b and in the light in the wavelength band below 580ηηι are turned on. O58 Finoleta 55a is installed.

 By locating the light source above the light-emitting end, light emission is prevented from directly hitting the finolators. This is in view of the purpose of preventing scattered light or stray light from being generated from an edge of a filter or the like, thereby causing an obstacle to light reception.

 Further, it is conceivable to configure a switching filter in the lens unit 3, but it is a difficult point that light must be shielded, resulting in an increase in cost. Further, as described above, there is a disadvantage in the case where the lens is configured to be replaceable. FIG. 11 shows a perspective view of a filter moving section (filter moving means) 23. The filter moving unit 23 includes a driving unit 90 for driving the support frame 94, the filter unit 55, and the stage 2 in parallel with the surface of the stage 2, and a driving force of the driving unit 90. "The drive port 9 1 for transmitting to the unit 5 5 and the panel 9 2 which is locked to the support frame 9 4 and the drive port 9 1 and urges the drive port 9 1 to the stage 2 side. Has been done.

The light source switching determines that the filter unit 55 needs to be moved. Then, the control means 81 gives a drive instruction for moving the filter unit 55 to the drive means 90 of the filter moving means. The driving means 90 drives the filter unit 55 to advance and retreat in parallel with the surface of the stage 2 via the driving port 91.

 Guide holes 93 are formed in the filter unit 55 to guide the movement so as to guide the movement direction and the position so as not to deviate from the prescribed positions. FIG. 11 shows a state in which the O 580 filter 55 a that focuses on a wavelength of 580 nm or less has moved to the imaging position. When green light is emitted from the light source unit 4, the driving means 90 performs driving for movement, moves the filter unit 55 in the direction of the arrow A, and moves the filter unit 55 in a wavelength band lower than 420 11 m. Move the L42 filter 55b that cuts light to the imaging position.

 By the way, the focal length of the lens 3a changes depending on the wavelength. For example, the UV light received by the L42 filter 55b, which cuts the wavelength in the band below 420nm, is the O58 filter, which cuts the wavelength band where the focal length of the lens 3a is 580nm or less. 5 Shorter than the focal length of the green light received at 5a.

 Therefore, the light received by the L42 finoletor 55b, which cuts the wavelength band below 420 nm, has a focal position before the CCD96 (see FIG. 12). The image when this light reaches the CCD 96 is in a so-called out-of-focus state. Therefore, in order to properly adjust the focal position, it is necessary to adjust (exchange) the position of the lens 3a, adjust the position of the CCD 96, or adjust the position of the object.

 Furthermore, the image pickup device 1 is an enlarged optical system, and the contour of an individual cannot be clearly extracted unless the depth of focus is strictly observed. Since it is a square-radiation exposure system, there is little background noise, and even if edge-enhanced image processing is performed, the original image quality is required sufficiently.

 Therefore, in the present invention, an optical path length adjusting means 95 for adjusting the optical path length is provided in one of the filters of the filter unit 55 for adjusting the focal length.

 FIG. 12 shows a configuration diagram of the sample 99, the filter unit 55, and the lens means (lens unit) 3.

In FIG. 12, ( _a ) shows that the lens 3a is focused through a filter 55b (in the case of green light, the filter 55a) to receive the fluorescence generated by the subject 99. FIG. The focal position X is the fluorescent light (6 (17 nm), the focal position coincides with the light receiving surface of the CCD 96 at the focal position. The focal position Y is the focal position when the fluorescent light (461 nm) of the subject 99 irradiated with the ultraviolet light is received, and the focal position Y is in front of the light receiving surface of the CCD 96. As shown in Fig. 12 (b), the difference in optical path length (chromatic aberration) is properly compensated for even when the irradiation light is changed, as shown in Fig. 12 (b), in order to resolve the shift of the focal position due to the difference in the wavelength of the light from the specimen. In order to adjust the optical path length, there is provided an optical path length adjusting means that can be replaced by a member separate from the filter.

 In the example shown in FIG. 12 (b), when ultraviolet fluorescent light (461 nm) is received, the optical path length adjusting member 95 is placed on the filter 55b. In this example, the optical path length adjusting member 95 is an adjusting glass 95.

 The fluorescence of the subject 99 is refracted by the adjusting glass 95, and the optical path length is adjusted. With this adjustment, the focal position of the image passing through the lens 3a matches the light receiving surface of the CD 96. With the adjusting glass 95, the optical path length is adjusted, so that it is possible to obtain precise light having the same focal length even for light in different wavelength regions from the subject.

 As described above, the optical path length adjusting means adjusts the focal position by superimposing the adjusting glass 95 on a predetermined filter. Then, the movement of the filter unit 55 is performed by the filter moving unit 23 according to an instruction from the control unit 81 based on the information on the exposure unit, the irradiation unit, and the information on the current filter type.

 Further, in the imaging apparatus 1 according to the present invention, when mounting the subject 99, the stage 2 is moved to the front cover 5 open position, the front cover 5 (shown in FIG. 2) is opened, and the subject 99 is mounted. In addition, since the filter can be operated from above the filter unit 55 at the time of maintenance of the apparatus, even if dust adheres to the filter, maintenance can be easily performed.

 In addition, a filter unit 55 is provided in the space between the stage 2 and the lens unit 3, and the filter is positioned above the light emitting position of the light source. There is no need to increase it. Furthermore, it can be said that the configuration is optically stable.

Supplementally, in the explanation, there is a treatment of the positional relationship of upward and downward, but this is for convenience. It goes without saying that the same configuration is possible when the device is mounted diagonally or configured horizontally.

 Thus, the imaging apparatus according to the present invention makes it possible to adjust, with a simple mechanism, different optical path lengths in a wavelength region in imaging weak fluorescence from an object having a plurality of different wavelength regions. It was possible to image minute specimens precisely without increasing noise light.

 In addition, by configuring the filter at such a position, it was possible to easily realize readjustment of the optical path length even if the filter was replaced to change the magnification of the lens.

 Here, the imaging operation of the present apparatus will be described based on the flowchart shown in FIG. When the switch 10 of the device is turned on and the power is turned on (Sl), the CPU of the main body performs an initialization operation for starting imaging (S2). When the door (front cover 5) is opened, a signal to open the door is issued (S3). At the start of reading, the stage 2 moves to the front, which is the open position of the front cover 5, based on a movement command from the data processing device 100 receiving this signal.

 The sample (subject) 99 of the emblem kit 40 or the fiem / rem kit 41 is mounted on the stage 2 (S4). When the front cover 5 is closed by the operator's hand (S5), the stage 2 moves to the origin (home position, which is the initial position of the photographing position) (S6). The signal of the cover sensor is sent to the data processing device 100 connected to the interface, and the preparation is displayed on the display.

 Here, the operator instructs the data processing device 100 to move the stage 2 to the measurement position (S7). Switch the LED filter (S8). A measurement start command is sent to the imaging device to start measurement (S9), and data is taken in (S10). When the data measurement continues, the designated data processing device sends a measurement start command to the imaging device (S12).

The control unit of the imaging device moves the stage 2 to the home position. S7 to S12 are repeated until the measurement is completed. When the measurement is completed, an initialization operation for completing the measurement is performed (S13). The stage 2 moves to the front where the front cover 5 is opened, opens the door (S14), and takes out the sampling sheet (S15). Here, when there is a next sampling sheet, the operations from S4 to S16 are repeated. If there is no next sampling sheet, close the door (S17), turn off the power and finish (S18, S19) o

 The imaging aperture is opened so that the light beam from the light converging means is irradiated on the entire area to be read on the stage. Thus, light is applied to the entire area to be read in.

 Further, in the present invention, by setting the distance between the condenser lens and the sampling kit within a certain focus depth range, the sampling corresponding to a plurality of types of sampling kits can be performed without installing an expensive automatic focusing device. A kit placement device was realized. Hereinafter, the subject of the individual number counting system of the present invention will be described in detail, but before that, the photoelectric conversion means (CCD966) itself, which is the control target, will be briefly described. Note that, here, the CCD 96 is described as an example of the photoelectric conversion means, but the invention can be applied to photoelectric conversion elements of the MS type or other types similarly.

 As described in Figure 14, photo sensors (such as photodiodes) are two-dimensionally arranged in a package. The rooster area has transparent windows to allow light to enter. Terminals VDD and GND apply the basic voltage. Extract video signal There are ク ロ ッ ク ψ 1 and Η φ 2 terminals for clocks synchronized with horizontal video signal transfer from the VOUT terminal.

 Vφ1, Vφ2, νφ3, νφ4 are vertical synchronization clocks for repeating horizontal synchronization in the vertical direction, and operate in four phases. The φRG terminal is a reset gate that controls the exposure time of one screen. The φ SUB terminal is a clock terminal for clearing a small amount of charge that is internally accumulated due to photoelectric conversion by exposure and remains after passing by the transfer clock.

 In this way, by controlling the clock applied to each terminal of the photoelectric conversion means, the exposure time and the output extraction are controlled.

The exposure control means transmits exposure instruction data controlled for each rod light source to the irradiation means 83 based on the instruction data received from the control means 81. Exposure control means 8 2 At least the first exposure time, which is longer than the minimum time for the individual to emit fluorescent light, and the second exposure time, which is the time when the photoelectric conversion means generates a pixel having an abnormal value due to long-time exposure, is set. The third time to perform is instructed to the irradiation means 83.

 The irradiating means 83, which has received the exposure data, irradiates the specimen on the stage with light of a specific wavelength region for a predetermined exposure time and for a predetermined number of times. At this time, the control means 81 adjusts the exposure time by controlling the transfer clock and the Z or reset pulse of the photoelectric conversion means 84.

Further, the irradiating means 83 has a light source control having a first mode in which the light source is turned on and exposed during multiple exposures according to the exposure time, and a second mode in which the light source is turned off and exposed. The control means includes subtraction means for subtracting the image data acquired in the second mode from the image data acquired in the first mode. The first method for counting the number of individuals contained in a sample is performed as follows. The fluorescence data of the individual (bacteria) in the specimen on the stage 2 that emits light by the irradiation light is transmitted to the photoelectric conversion means 84. Frame memory photoelectric converter 8 4 transmits to AZD converter ⁸ 5 to convert the video signal received binary image data, A Roh D Compur motor 8 5 digital converted data as the measurement data 8 Send to 6.

 When the imaging by the irradiation from the light source is completed, the frame memory 86 transmits the stored data to the control means 81. The counting means counts the number of individuals in the specimen that emit fluorescent light of a main wavelength different from the irradiated light of the specific wavelength region. Then, the adding means counts the number of individuals included in the specimen by cumulatively adding the image data at the same pixel position of the photoelectric conversion means.

 The imaging device 1 includes an interface means 80 for transmitting image data, and an external data processing apparatus 100 connected via the interface means 80 to form the control means 81. The processing is configured to be performed in cooperation with the data processing device 100. Therefore, both the adding means and the counting means may be on the imaging device 1 side having the photoelectric conversion element 96, or the data processing apparatus 1 which controls the imaging apparatus 1 having the photoelectric conversion element 96. It may be on the 0 side.

Furthermore, they may be realized by hardware or software, and The software may be provided on a recording medium, or may be provided in a form of download provided by communication or the like. Alternatively, the adding means and / or the counting means may exist and be connected as independent units, or may be provided as a form such as an AS IC or a mask ROM.

 Here, a second example of the method of counting individuals (bacteria) included in a specimen according to the present invention will be described.

 As shown in the flowchart of FIG. 15, the point is to find the background level when subtracting the background value from each image data.

 The control means captures one shot of a partial area (for example, 20 dots X 20 dots) of the entire sample with a predetermined exposure time, for example, 2 seconds.

 Then, the pixel value hierarchy dividing means divides the received image data into a plurality of pixel hierarchies. The pixel value level (8Bit: 0 to 255) is divided into, for example, four sections of 0 to 63, 64 to: L27, 128 to 191 and 192 to 255.

 The background position specifying means records the pixel positions that fall into the 0 to 63 sections. For example, coordinates (5, 3), (7, 10), (11, 12).

 The record may be all or about five points, but here, three points are P1, P2, and P3. This is the representative of the three backgrounds.

 Here, the background position calculation means resets the CCD and starts again. In the above, the value after 2 seconds was fixed and recorded in the memory. However, this time, the AZD conversion is continuously and repeatedly recorded focusing on only the three points Pl, P2, and P3. For example, it is performed in units of 1 O Oms.

 Next, we want a level of 2.0 seconds, so the sum of the levels in each of the five seconds, 1.8, 1.9, 2.0, 2.1, and 2.2, e (1.8) Dividing + e (1.9) + e (2.0) + e (2.1) + e (2.2) by 5 gives a level of 2.0 seconds with white noise canceled. .

 Further, the same processing as the above-described five-point row is performed at three points Pl, P2, and P3, and an average value of three points is calculated. This value is defined as Bk. This is specified as a background pixel value hierarchy indicating the individual background on the subject.

Then, the value of each pixel of the whole image of one shot with 2 seconds exposure acquired earlier, LV (X, Y): When X and Y are the coordinates of the area, calculate LV (X, Y) one Bk for all X and Y. This entire data is transmitted to the data processing device via the interface as imaging data obtained by irradiation with ultraviolet light.

 Further, similarly, green light irradiation imaging is performed, and transmitted to the data processing device via the interface as image data acquired with green light.

 The data processor uses these two frame images to count bacteria and calculate viable bacteria.

 The imaging device 1 is connected to an interface means 80 for transmitting image data, and an external data processing device via the interface means 80, and the processing by each means constituting the control means 81 includes: It is configured to be performed in cooperation with the data processing device.

 Therefore, any of the pixel value hierarchy division means, the background pixel hierarchy division means, the background position calculation means, and the counting means may be provided on the imaging device side having the photoelectric conversion element 96 or the photoelectric conversion element 96 may be provided. It may be on the side of the data processing device that controls the imaging device provided.

 Further, they may be realized by hardware or software, and the software may be provided by a recording medium, or may be provided by download through communication or the like. Alternatively, any of these pixel value hierarchy division means, background pixel hierarchy division means, background position calculation means, and counting means may exist and be connected as independent units, or may be in the form of an ASIC or a mask ROM. It may be provided.

 The present invention also makes it possible to emphasize the number of individuals of the subject by utilizing the characteristic shown in FIG. 24.

 Hereinafter, acquisition of pixel data excluding the background level and the bright spot in the present invention will be described with reference to FIGS. 16 to 22.

Figure 16 is a graph of ideal subject (bacteria image) level exposure time. However, in reality, the background level around the subject is exposed as shown in FIG. 20, so that the background level acts greatly like the line of “bacterium + background” level in FIG. In other words, the system can be used to irradiate batteries with ultraviolet or green light. Is a system that obtains the excitation energy to generate fluorescence, and the system tries to acquire the fluorescence. Therefore, not only the light reception level is low, but also the contrast between the background and the subject is low.

 In order to capture a subject under such adverse conditions, it is necessary to perform open exposure for a certain period of time, for example, to take a picture of a constellation, much longer than the available exposure time of the CCD96.

However, when the CCD 96 is used in a time zone exceeding the usable time due to a defect in the manufacturing process, some pixels exhibit abnormal values called “bright spots” in the pixels. FIG. 18 shows this appearance state, in which the appearance frequency of the bright spot increases from the time _exceeding the predetermined time TMT is graphed.

The fluorescence onset time T _s shown in FIG. 16 is a predetermined time during which light of a specific wavelength given to a large molecule such as a bacterium accumulates energy, and fluorescence is generated after this T _s . Therefore, the time period t which can expose (the CCD 96) without generating in abnormality signal to the brightness as seen from FIG. 16 and FIG. 17, the usable range shown in FIG. 17, "T _S rather t < T _LMT .

19, above described "T _S rather 1 st frame exposure for each time satisfying t <T _LMT j, 2n d frame, 3 a diagram is repeatedly rd frame white level of bacteria in, respectively it Have reached V1, V2 and V3.

 Since the CCD is guaranteed for the amount of light input to each pixel and the clarity of the output voltage, it is possible to perform a line meter calculation, which is equivalent to the result of continuous exposure by calculating V1 + V2 + V3. And an image with no bright spots. When an image without luminescent spots generated in this manner is subjected to binary elimination using a predetermined value experimentally obtained in advance, only the bacterial portion can be recognized as white. If binarization is performed while including the bright spots, the bright spots will be mistakenly recognized as tactics.

 Therefore, it is extremely important to binarize images acquired with an exposure time within the range where no bright spots are generated, after superimposing the images several times until a desired level is reached.

In the above description, it is described that the individual is detected by binarization.However, the individual can be raised by filtering such as contour emphasis, etc. The detection processing algorithm for performing You will consider the best method.

 Now, since the brightness of the fluorescent light is small, it does not have sufficient contrast with the reflected light level of the background, and in fact the background output shown in Fig. 20 overlaps, and white noise peculiar to the semiconductor is superimposed. ing. For this purpose, a level for performing the above-described binarization must be set between the background value and the target value.

Therefore, the black level is excluded from the video levels V1, V2, and V3 obtained by dividing into frames. At this time, as shown in Fig. 21, the black level B _F is obtained for each frame as B _F or B _F2 , B _F3 , and VI- B _F V2—B _F2 or V3—B _F3 to obtain a beautiful image. But it's actually different.

White noise has a random cycle and cannot be canceled by image data processing. For this reason, the result of the above subtraction may rather increase the noise width (in some cases, the negative and negative and the positive and the positive may be deviated). Therefore, the address of the level N _w ho Wa Itonoizu, it is preferable treated separately phase and deal with the black level B _F.

For example, as shown in FIG. 20, the weight of N _w is set to 1 Z 3 by using the value of the black level B tota 1 exposed at T 1 + T 2 + T 3 hours as V1 + V2 + V3—B total as shown in FIG. Can be reduced.

In addition, using the property that the black level rises to the red, the AZD conversion is repeatedly performed with the exposure (sampling) before and after the target time T + T ₂ + T ₃ o'clock, and the number before and after digitally obtained is, for example, 10 points. By averaging the values, it can be approximated to the value of the center of B tota 1. Specifically, assuming that T1 + T2 + T3 uses 10 points before and after every 2ms in 2 seconds, 1.90, 1.91, 1.92, ... 1.9.9 9, 2, 2.01, 2.02, ··· The values that were A / D converted at 21 points in 2.1 are stored in the memory, and these values, ₉₀ , V _{x .91} , V ₂ .

 2.1

i ^ T upper ∑ 1 QV ₍ Or,

 [Equation 2]

1 dish (Vl Q ~ V2.1) ^{+ min} (Vi.9 ~ V2.1)

 It is also possible to set it to 2.

Open B _F * except N _w obtained in this way,

V “ _BF *, V ₂ — _BF *, V ₃ — _BF *

And

Furthermore, by the processing of the same idea as when this B _F * was obtained,

 [Equation 3]

Vi = (VI-BF), V 2 = (V 2 - B F), V 3 = (V 3 _B F)

, And the final output value Vout for image processing is

 [Equation 4]

V out = V 1 + V 2 ⁺ V 3

Is obtained. In the above description, the same pixel is sampled multiple times by exposure at different times, and background removal and noise cancellation are performed using the pixel values of the multiple times. Therefore, it is possible to use a plurality of neighboring points without making the pixel position of the operation value acquisition target strictly the same.

In Figure 21, B _Fi is black level in the i-th frame, always white noise ± N _W regardless of the exposure is superimposed.

 In FIGS. 20 to 22, each frame time T1, T2, T3 (assuming continuous exposure start) includes a fluorescence start time Ts (must be longer than Ts, Tl> Ts, T2> Ts, T3> Ts).

As described in detail above, in the present invention, the range of the bright spot noise is not generated. Since the image data of the subject from multiple exposure times is added, the occurrence of luminance noise (bright spots) in the CCD 96 is eliminated, and the number of individuals included in the measured object with low light intensity can be saved. It was done.

 FIG. 23 (a) shows an integrated device in which all of the imaging means, the adding means, and the counting means (individual counting means) are included. Fig. 23 (b) shows a form in which an imaging device is interfaced to a combi- ter, and the individual is counted by the combi- ter.

 Fig. 23 (c) shows a case where the addition means is unitized and only the accumulation function is independent. This is the case where the computer is sent to the computer and the individual power is counted on the computer side.

 Fig. 23 (d) shows a form in which a normal camera is connected to a computer, and is an example in which accumulation processing and individual count processing are performed in the computer. In this case, it is often realized by software (program), but it is also possible to convert it to hardware and mount it on an option board. This program may already exist in the computer, or may be recorded on a storage medium such as a CDRQM as a product attached to the imaging device.

 Fig. 23 (e) is a networked case where the cable between the camera and the computer in Fig. 23 (d) is a cable, and this network is not limited to wired, wired and wireless. Can take a wide range of forms, LAN, Internet. Further, the CCD 96 has been described as an example of the photoelectric conversion means, but the present invention may be a MOS type or other type of photoelectric conversion element. Industrial applicability

 INDUSTRIAL APPLICABILITY The present invention is applicable to an imaging apparatus for imaging a minute individual that is excited by irradiation light in a plurality of different wavelength regions and generates light having a wavelength different from the wavelength region of the irradiation light, and converts the light into an electric signal. It is possible.

Claims

The scope of the claims

1. An imaging device that captures an image of a minute subject that emits light in a wavelength region different from this wavelength region according to irradiation light in at least two wavelength regions and converts the image into an electric signal. The stage to load,

 Light source means for illuminating a surface of the stage loaded with the subject,

 Light switching means for switching the irradiation light emitted by the light source means to light rays of a plurality of different wavelength ranges,

 Lens means for condensing light from the subject illuminated by the plurality of different wavelength regions;

 Photoelectric conversion means for converting the two-dimensional image light collected by the lens means into an electric signal,

 And two or more filter units that are switched according to switching of light beams by the light switching unit,

The imaging apparatus according to claim 1, wherein the filter unit is disposed in an optical path between the lens unit and a position where the light source unit emits light.

 2. The imaging apparatus according to claim 1, further comprising a moving unit that supports the two or more filter units and moves the filter units in a direction intersecting a light receiving direction of the lens unit.

 3. The imaging device according to claim 1, wherein the two or more filter units are made of materials having different thicknesses or different refractive indexes.

 4. An optical path length adjusting means provided on at least one of the two or more filter means and adjusting an optical path length between the stage and the photoelectric conversion means. An imaging device according to claim 1.

 5. The imaging device according to claim 1, further comprising an exposure control unit that controls an exposure time of the photoelectric conversion unit.

 6. A stage on which a collection kit containing the individual to be imaged is loaded;

Fixing means, which is provided on the stage so as to be openable and closable, and which fixes the collection kit and has an imaging opening; A light converging means for converging light from the light emitting means and projecting the light as a light beam on the stage; and a light source means for irradiating the stage with light. Lens means for condensing light from the individual;

 Photoelectric conversion means for converting the light condensed by the lens means into an electric signal; and

 The imaging opening is so opened that the light beam emitted from the light converging means is applied to the entire region to be read on the stage.

7. The light emitting means is constituted by a plurality of light emitting devices, and is opposed to the center of the lens of the lens means so that light beams from the individual light emitting devices are projected to substantially the same position on the stage. It is located at a symmetrical position that is the distance,

 The imaging opening is a polygon having an apex angle as an opening that allows light to pass in the light projection direction of the plurality of light emitting units.

7. The imaging device according to claim 6, wherein: ,

 8. The stage is formed so that a transparent or translucent sheet-shaped first collection kit or a second collection kit having an opaque background is selectively loaded, and the fixing means includes:

 A supporter for supporting the mounting member on which the first collection kit is mounted and which reflects the light emitted from the light source means to the individual and has an opaque background;

 7. The imaging device according to claim 6, further comprising: an elastic means for fixing the first collection kit in cooperation with the placing member.

9. Moving means for moving horizontally while maintaining the height of the stage surface of the stage with respect to the lens means,

 A first shortest distance from the lens unit when the first collection kit is placed is a second shortest distance from the lens unit when the second collection kit is placed on the stage. 9. The imaging device according to claim 8, wherein the thickness of the placement member is set so as to be equidistant from the imaging member.

10. A stage for loading a subject including an individual to be imaged, Light source means for irradiating a light beam onto the stage surface of the stage; lens means for condensing light from the subject irradiated by the light source means; and two-dimensional image light condensed by the lens means And photoelectric conversion means for converting into a signal.

 The light source means,

 A plurality of light means for irradiating the stage surface with light from an obliquely upward direction; and the plurality of light means so that the light emitted from the plurality of light means irradiates substantially the same position on the stage surface. Holder means for supporting the light beam means,

 An image pickup apparatus comprising: fixing means for integrally fixing the holder means and the lens means.

1 1. The light beam means

 A first light means comprising light means for emitting light in a first wavelength region, and a second light means comprising light means for emitting light in a second wavelength region. Item 10. The imaging device according to Item 10.

 12. Each of the first beam means and the second beam means further comprises a plurality of beam means for irradiating the stage surface with light beams from a plurality of directions symmetrical to the lens means. The imaging device according to claim 11, wherein the imaging device is configured.

1 3. The light beam means

 A light-emitting element provided at one end of a hollow case having a mouth shape,

 A lens unit provided inside the hollow case and / or at another end of the hollow case, for converging a light beam from the light emitting element;

 Filter means for controlling a wavelength region transmitted through the lens,

The imaging device according to claim 10, wherein the imaging device is configured by:

14. The holder according to claim 10, wherein the holder has a positioning portion for positioning the light beam at a predetermined mounting position of the holder.

15. The fixing means is configured to movably support the lens means corresponding to the substantially same position on the stage surface irradiated by the plurality of light beam means. The imaging device according to claim 10, wherein: