JP4979777B2 - Microscope image processing device - Google Patents

Description

  The present invention relates to a microscope image processing apparatus capable of freely selecting wavelengths of a plurality of lights, independently adjusting the light intensity, and simultaneously irradiating a sample with these lights.

  In general, fluorescent microscopes are widely used for the purpose of detecting proteins, genes, and the like that have been subjected to fluorescent labeling of biological tissues and cells in medical and biology and other fields. In recent years, in particular, multiple fluorescence detection, in which specimens stained with multiple fluorescent dyes or specimens expressing multiple fluorescent proteins are observed at once in time, is very useful for analyzing genes and elucidating intracellular structures. ing. In the multiple fluorescence detection, means for irradiating the sample with irradiation light with a plurality of wavelengths is widely used to excite the multiple fluorescence sample. In this case, the time interval for irradiating each wavelength is short, The fact that the intensity distribution of irradiation light for each wavelength on the sample surface does not change temporally and spatially is an important factor that affects the accuracy of observation data.

  Conventionally, as a means for irradiating a specimen with irradiation light having a plurality of wavelengths for exciting a multiple fluorescent specimen, an apparatus that uses a filter switching means to time-divide the wavelength of the excitation light (see, for example, Patent Document 1). An apparatus using two independent light sources (see, for example, Patent Document 2) has been proposed. Conventionally, an apparatus (for example, Patent Document 3) that divides light from a single light source and irradiates a specimen has been proposed.

  As shown in FIG. 11, the apparatus described in Patent Document 1 previously processes a living tissue to be measured by a plurality of fluorescence processes, and a plurality of excitation light selection filters 151 a are arranged at predetermined locations in one rotating disk 151 b. The filter switching means 151 installed in the filter and the filter switching means 152 installed in other places in the rotary disk 152b with the plurality of fluorescence selection filters 152a are rotated in synchronization with each other, thereby the first The biological tissue is irradiated with the excitation light and the second excitation light in a time-sharing manner, and the first fluorescence and the second fluorescence generated from the biological tissue are sequentially recorded. For example, the change in intracellular ion concentration and the membrane potential It is configured to measure each change substantially simultaneously.

  Further, as shown in FIG. 12, the apparatus described in Patent Document 2 uses two white light sources 161A and 161B such as a xenon lamp, and condenses light from each lamp via collector lenses 162A and 162B. Thereafter, the wavelength is selected by transmitting the excitation filters 163A and 163B having different transmission wavelength ranges, and the light of the selected wavelength is synthesized by the dichroic mirror 164 and introduced into the observation optical system 165. . According to the apparatus described in Patent Document 2, it is possible to illuminate at a desired wavelength by appropriately exchanging the excitation filters 163A and 163B.

  Further, as shown in FIG. 13, the apparatus described in Patent Document 3 divides the irradiation light emitted from the irradiation light source 171 via the branch optical system 172, and separates the lights A and B on the sample 173. It is comprised so that it may irradiate to different site | parts.

JP 09-005243 A Japanese Patent Application Laid-Open No. 07-056092 Japanese Patent Laid-Open No. 10-090608

  However, in the apparatus described in Patent Document 1, the filter for selecting the first excitation light and the filter for selecting the second excitation light or the filter for selecting the first fluorescence and the filter for selecting the second fluorescence are rotated. There is a slight time loss due to switching, and this time loss is unsuitable for measuring fast-moving samples such as nematodes and fast-changing phenomena such as propagation of calcium ion concentration in the myocardium. is there.

  As a means for driving such a rotating disk, a motor or the like is generally used, but there arises a problem that the focus is shifted due to the influence of vibration generated by the motor. Therefore, in order to reduce the vibration generated when the rotating disk is driven, it is necessary to take a measure such as placing the microscope set on a vibration isolation table constituted by an air spring or the like.

  As a method that does not have a mechanical drive unit such as a rotating disk, an interference filter (excitation filter) that has a peak transmittance at a plurality of specific wavelengths is used. A method of simultaneously irradiating the two is considered. According to this method, since there is no mechanical drive unit, problems such as generation of vibration do not occur. However, it is difficult to modify the transmission characteristics of an excitation filter manufactured to have such characteristics after manufacturing. Thus, for example, when measuring a sample stained with two different dyes, if the fluorescence corresponding to one dye is strong and the fluorescence corresponding to the other dye is weak, the wavelength corresponding to each dye is weak. There is a problem that the intensity of the excitation light cannot be adjusted.

  In the apparatus described in Patent Document 1, a dichroic mirror having high reflection characteristics only at two or three specific wavelengths is used as the dichroic mirror 158 that guides excitation light to the sample 157. Generally, the dichroic mirror is first created, and then the excitation filter is designed and manufactured according to the reflection characteristics of the dichroic mirror. The reflection peak wavelength of the dichroic mirror and the transmission peak wavelength of the excitation filter are perfectly matched. This is difficult, and as a result, there is a problem that the cost becomes very expensive.

  Further, in the apparatus described in Patent Document 2, since the intensity of the light source varies randomly for each of the two light sources, the ratio value of the fluorescence image excited by the light from each light source is calculated by this variation. Has the problem of becoming uncertain.

  The apparatus described in Patent Document 3 is configured to divide light from a light source having a single wavelength and irradiate a plurality of different parts on the sample. Therefore, the sample cannot be irradiated with light having a plurality of wavelengths with the same irradiation intensity distribution.

The present invention has been made to solve the above-mentioned problems, and can irradiate a sample with light of a plurality of wavelengths at the same time and with the same irradiation intensity distribution. The wavelength and intensity of each light are independent. It is an object of the present invention to provide an inexpensive microscope image processing apparatus that can be set.

To achieve the above object, the microscope image processing apparatus according to the onset Ming, a white light source, the irradiation light beam splitting means for splitting the light flux of irradiation light emitted from the white light source into a plurality of light beams, split an irradiation light wavelength selecting means for selecting a wavelength of the illumination light passing through each of the optical path respectively provided on the optical path of the irradiation light, the optical path respectively provided on the optical path of the split each of the irradiation light an irradiation light quantity adjusting means for adjusting the light quantity of the illumination light passing through the irradiated light beam combining means for combining a plurality of light beams of the irradiation light quantity wavelength is selected is adjusted to a single light beam, synthesis a mirror that transmits the fluorescence of the irradiation light from the specimen excited by該照Shako guides in a direction to irradiate the sample which is, an objective lens disposed between said mirror sample, the objective Len And a fluorescent light beam splitting means for splitting the fluorescent light flux that has passed through the mirror into a plurality of light fluxes, and a fluorescence that is provided on each of the divided light paths of the fluorescence and that selects the wavelength of the fluorescence that passes through the light path a wavelength selection means, and an imaging means you capture a fluorescent image by the fluorescent light passing through the optical path provided on an optical path of the fluorescence wavelength is selected, the combining process a plurality of the fluorescent image is an image shooting Image processing means and image display means for displaying the combined fluorescent image, and the irradiation light amount adjusting means adjusts the amount of the irradiation light individually passing through the irradiation. has become possible to adjust the intensity of light, the fluorescence intensity of each of the fluorescence wavelength is selected using at least one of the irradiation light quantity adjusting means and the image processing means the Characterized in that it in.
In order to achieve the above object, a microscope image processing apparatus according to the present invention includes a white light source, a light beam of irradiation light emitted from the white light source, a light beam of first irradiation light, and a light beam of second irradiation light. Irradiating light beam splitting means for splitting the first irradiating light, first irradiating light wavelength selecting means for selecting a wavelength of the first irradiating light provided on the optical path of the first irradiating light, and A first irradiating light amount adjusting means for adjusting a light amount of the first irradiating light provided on the optical path; and a first one for selecting a wavelength of the second irradiating light provided on the optical path of the second irradiating light. The second irradiation light wavelength selection means, the second irradiation light quantity adjustment means provided on the optical path of the second irradiation light to adjust the light quantity of the second irradiation light, and the wavelength is selected and the light quantity is adjusted. The light beam of the first irradiation light and the light beam of the second irradiation light whose wavelength is selected and the amount of light is adjusted. Irradiating light beam combining means for combining into one light beam, a mirror for guiding the combined irradiation light in the direction of irradiating the sample and transmitting fluorescence from the sample excited by the irradiation light, the mirror and the mirror An objective lens arranged between the sample and the fluorescent light beam splitting means for splitting the fluorescent light beam transmitted through the objective lens and the mirror into a first fluorescent light beam and a second fluorescent light beam; A first fluorescence wavelength selecting means provided on the optical path of the first fluorescence to select the wavelength of the first fluorescence; and a wavelength of the second fluorescence provided on the optical path of the second fluorescence. The second fluorescence wavelength selection means, the first imaging means provided on the optical path of the first fluorescence for which the wavelength is selected, and the first fluorescence image by the first fluorescence, and the wavelength are selected. Provided on the optical path of the second fluorescence. The second imaging means for picking up the second fluorescent image by the image, the image processing means for combining the first fluorescent image and the second fluorescent image, and the image for displaying the combined fluorescent image Display means, and the first irradiation light intensity adjustment means and the second irradiation light intensity adjustment means adjust the light intensity of the irradiation light that individually passes through the intensity of the irradiation light. The intensity of the first fluorescent light can be adjusted using at least one of the first irradiation light quantity adjustment means, the second irradiation light quantity adjustment means, and the image processing means. And the fluorescence intensity of the second fluorescence are made equal to each other.
In order to achieve the above object, a microscope image processing apparatus according to the present invention is divided into a white light source, and an irradiation light beam splitting unit that splits a light beam of irradiation light emitted from the white light source into a plurality of light beams. Irradiation light wavelength selection means for selecting the wavelength of the irradiation light that is respectively provided on the optical path of the irradiation light and that passes through the optical path; and And an irradiation light beam adjusting unit that adjusts the light amount of the irradiation light that passes through, and an irradiation light beam combining unit that combines a plurality of light beams of the irradiation light whose wavelength is selected and the light amount is adjusted into a single light beam. A first objective lens that guides the irradiation light in a direction to irradiate the sample, and the sample that is disposed opposite to the first objective lens across the sample and excited by the synthesized irradiation light From A second objective lens on which fluorescence is incident; a fluorescent light beam splitting means for splitting the fluorescent light beam incident on the second objective lens into a plurality of light beams; and provided on each of the divided optical paths of the fluorescence. A fluorescence wavelength selection unit that selects a wavelength of the fluorescence that passes through the optical path, an imaging unit that is provided on the optical path of the fluorescence whose wavelength is selected, and that captures a fluorescence image of the fluorescence that passes through the optical path; Image processing means for synthesizing the plurality of fluorescent images that have been synthesized, and image display means for displaying the synthesized fluorescence images, and the irradiation light quantity adjusting means is individually radiated through It is possible to adjust the intensity of the irradiation light by adjusting the amount of light, and the wavelength is selected using at least one of the irradiation light amount adjustment means and the image processing means. The fluorescence intensity of each of the fluorescent, characterized in that to equalize.
In order to achieve the above object, a microscope image processing apparatus according to the present invention includes a white light source, a light beam of irradiation light emitted from the white light source, a light beam of first irradiation light, and a light beam of second irradiation light. Irradiating light beam splitting means for splitting the first irradiating light, first irradiating light wavelength selecting means for selecting a wavelength of the first irradiating light provided on the optical path of the first irradiating light, and A first irradiating light amount adjusting means for adjusting a light amount of the first irradiating light provided on the optical path; and a first one for selecting a wavelength of the second irradiating light provided on the optical path of the second irradiating light. The second irradiation light wavelength selection means, the second irradiation light quantity adjustment means provided on the optical path of the second irradiation light to adjust the light quantity of the second irradiation light, and the wavelength is selected and the light quantity is adjusted. The light beam of the first irradiation light and the light beam of the second irradiation light whose wavelength is selected and the amount of light is adjusted. An irradiation light beam synthesizing means for synthesizing into one light beam, a first objective lens for guiding the synthesized irradiation light in the direction of irradiating the sample, and an arrangement facing the first objective lens across the sample And the second objective lens into which the fluorescence from the sample excited by the synthesized irradiation light is incident, and the fluorescent luminous flux incident on the second objective lens is converted into the first fluorescent luminous flux and the first fluorescent luminous flux. Fluorescent light beam splitting means for splitting into two fluorescent light fluxes, first fluorescence wavelength selection means for selecting a wavelength of the first fluorescence provided on the optical path of the first fluorescence, and the second fluorescence A second fluorescence wavelength selection means for selecting the wavelength of the second fluorescence provided on the optical path of the first fluorescence, and a first fluorescence by the first fluorescence provided on the optical path of the first fluorescence for which the wavelength is selected. A first image pickup means for picking up a fluorescent image, and the second of which the wavelength is selected. A second imaging unit that is provided on the optical path of the light and that captures a second fluorescent image by the second fluorescence; and an image processing unit that combines the first fluorescent image and the second fluorescent image. Image display means for displaying the synthesized fluorescent image, and the first irradiation light quantity adjustment means and the second irradiation light quantity adjustment means individually pass the irradiation light. It is possible to adjust the intensity of the irradiation light by adjusting the amount of light, and at least one of the first irradiation light amount adjustment unit, the second irradiation light amount adjustment unit, and the image processing unit. And using the same, the fluorescence intensity of the first fluorescence and the fluorescence intensity of the second fluorescence are made equal.
In the above-described microscope image processing apparatus, it is preferable that the irradiation light beam splitting unit and the irradiation light beam combining unit are dichroic mirrors.
In the microscope image processing apparatus, it is preferable that the fluorescent light beam splitting unit is a dichroic mirror.

In order to achieve the above object, a microscope image processing apparatus according to the present invention includes a white light source, a light beam of irradiation light emitted from the white light source, a light beam of first irradiation light, and a light beam of second irradiation light. Irradiating light beam splitting means for splitting the first irradiating light, first irradiating light wavelength selecting means for selecting a wavelength of the first irradiating light provided on the optical path of the first irradiating light, and A first irradiation light amount adjusting means for adjusting the light amount of the first irradiation light provided on the optical path; and a polarization direction of the first irradiation light provided on the optical path of the first irradiation light. A first irradiation light polarization unit; a second irradiation light wavelength selection unit which is provided on the optical path of the second irradiation light and selects a wavelength of the second irradiation light; and the light of the second irradiation light. A second irradiation light amount adjusting means provided on the road for adjusting the amount of the second irradiation light; and A second illuminating light polarization means for selecting a polarization direction of the second illuminating light, the transmission polarization axis being set perpendicularly to the first illuminating light polarizing means provided on the road; Irradiation light that combines the light beam of the first irradiation light that has been adjusted and the polarization direction selected and the second irradiation light beam that has been selected in wavelength and whose light amount has been adjusted and the polarization direction has been selected into a single light beam. A light beam synthesizing unit, a mirror that guides the synthesized irradiation light in a direction to irradiate the sample and transmits fluorescence from the sample excited by the irradiation light, and is disposed between the mirror and the sample An objective lens, a fluorescent light beam splitting means for splitting the fluorescent light beam transmitted through the objective lens and the mirror into a first fluorescent light beam and a second fluorescent light beam, and an optical path of the first fluorescent light The transmission polarization axis provided is the first illumination A first fluorescence polarization means set parallel to the polarization means, and a second fluorescence polarization provided on the optical path of the second fluorescence and having a transmission polarization axis set parallel to the second irradiation light polarization means Means, a first imaging means provided on the optical path of the first fluorescence transmitted through the first fluorescence polarization means, and taking a first fluorescence image by the first fluorescence, and the second fluorescence A second imaging means provided on the optical path of the second fluorescence transmitted through the polarization means to capture a second fluorescence image by the second fluorescence; the first fluorescence image; and the second fluorescence image. And image display means for displaying the fluorescent image that has been subjected to the synthesis process, and the first irradiation light quantity adjustment means and the second irradiation light quantity adjustment means. Adjusts the amount of the irradiation light that individually passes through each of the irradiation lights. It is possible to adjust the intensity, and at least one of the first irradiation light amount adjustment unit, the second irradiation light amount adjustment unit, and the image processing unit is used. The intensity is equal to the fluorescence intensity of the second fluorescence.
Furthermore, in order to achieve the above object, a microscope image processing apparatus according to the present invention includes a white light source, a light beam of irradiation light emitted from the white light source, a light beam of first irradiation light, and a light beam of second irradiation light. Irradiating light beam splitting means for splitting the first irradiating light, first irradiating light wavelength selecting means for selecting a wavelength of the first irradiating light provided on the optical path of the first irradiating light, and A first irradiation light amount adjusting means for adjusting the light amount of the first irradiation light provided on the optical path; and a polarization direction of the first irradiation light provided on the optical path of the first irradiation light. A first irradiation light polarization unit; a second irradiation light wavelength selection unit which is provided on the optical path of the second irradiation light and selects a wavelength of the second irradiation light; and the light of the second irradiation light. A second irradiation light amount adjusting means provided on the road for adjusting the amount of the second irradiation light; and the second irradiation light. A second illuminating light polarizing means which is provided on the optical path and whose transmission polarization axis is set perpendicular to the first illuminating light polarizing means and selects the polarization direction of the second illuminating light; Irradiating light that combines the light beam of the first irradiation light whose polarization is selected and the second irradiation light beam of which the wavelength is selected and the light amount is adjusted and the polarization direction is selected into a single light beam. By a light beam combining means, a first objective lens that guides the synthesized irradiation light in a direction to irradiate the sample, and the synthesized irradiation light that is disposed opposite to the first objective lens across the sample. A second objective lens into which the fluorescence from the excited sample is incident, and a fluorescence that divides the luminous flux incident on the second objective lens into a first fluorescent luminous flux and a second fluorescent luminous flux. A light beam splitting means and a transparent light path provided on the optical path of the first fluorescence; A first fluorescence polarization unit whose polarization axis is set parallel to the first irradiation light polarization unit, and a transmission polarization axis which is provided on the optical path of the second fluorescence and parallel to the second irradiation light polarization unit. A second fluorescence polarization unit set to 1 and a first fluorescence image provided on the optical path of the first fluorescence transmitted through the first fluorescence polarization unit and capturing a first fluorescence image by the first fluorescence An imaging unit; a second imaging unit that is provided on the optical path of the second fluorescence transmitted through the first fluorescence polarization unit; and that captures a second fluorescence image by the second fluorescence; and the first Image processing means for combining the fluorescent image and the second fluorescent image, and image display means for displaying the combined fluorescent image, the first irradiation light amount adjusting means, The second irradiation light amount adjustment means adjusts the amount of irradiation light that passes individually. Thus, the intensity of the irradiation light can be adjusted, and at least one of the first irradiation light amount adjustment unit, the second irradiation light amount adjustment unit, and the image processing unit is used. The fluorescence intensity of the first fluorescence is made equal to the fluorescence intensity of the second fluorescence.
In the above-described microscope image processing apparatus, the polarizing means is preferably a polarizing plate or a polarizing beam splitter.
In the microscope image processing apparatus, the image processing unit calculates a luminance ratio between the plurality of fluorescent images, and the image display unit displays the original image and the ratio image of the combined fluorescent image. It is preferable to do.

Furthermore, in the above-described microscope image processing apparatus, it is preferable that the irradiation light amount adjustment unit is insertable into and removable from the optical path of the irradiation light.

According to the present invention, an inexpensive microscope image processing apparatus that can irradiate a sample with light of a plurality of wavelengths simultaneously and with the same irradiation intensity distribution, and can independently set the wavelength and intensity of each light. Can be provided.

It is a schematic block diagram of the illuminating device of the fluorescence microscope concerning Example 1 of this invention, and the image processing apparatus using the illuminating device. It is a schematic block diagram which shows the modification of the illumination part of the fluorescence microscope concerning the modification of Example 1 of this invention. It is a schematic block diagram of the illuminating device of the fluorescence microscope concerning Example 2 of this invention, and the image processing apparatus using the illuminating device. It is a schematic block diagram of the illuminating device of the fluorescence microscope concerning the modification of Example 2 of this invention, and the image processing apparatus using the illuminating device. It is a schematic block diagram of the illuminating device of the fluorescence microscope concerning Example 3, and the image processing apparatus using the illuminating device. It is a schematic block diagram of the illuminating device of the fluorescence microscope concerning Example 4 of this invention, and the image processing apparatus using the illuminating device. It is a schematic block diagram of the illuminating device of the fluorescence microscope concerning Example 5 of this invention, and the image processing apparatus using the illuminating device. It is a schematic block diagram of the illuminating device of the fluorescence microscope concerning Example 6 of this invention, and the image processing apparatus using the illuminating device. It is a schematic block diagram of the illuminating device of the fluorescence microscope concerning Example 7 of this invention, and the image processing apparatus using the illuminating device. FIG. 10 is a schematic configuration diagram of an illumination device for a fluorescence microscope and an image processing device using the illumination device according to a modification of Example 7. It is a schematic block diagram which shows one means to irradiate the sample with the irradiation light by multiple wavelengths for exciting the conventional multiple fluorescence sample. It is a schematic block diagram which shows the other means to irradiate a sample with the irradiation light by multiple wavelengths for exciting the conventional multiple fluorescence sample. It is a schematic block diagram which shows the prior art example of the apparatus which divided | segmented the light from a single light source and irradiated a sample.

Prior to the description of the embodiments, the effects of the present invention will be described.
With the configuration as in this onset bright, only one light source, it is possible to illuminate the sample with light of different or two wavelength regions, as in the case of using a plurality of light sources, the space of each light source Uniform illumination without being affected by the difference in general intensity distribution.
In addition, since the sample can be excited with a plurality of different or two wavelength bands with only one light source, the influence of the temporal variation of each light source is affected as in the case of using a plurality of light sources. A ratio image can be obtained without fail.
In addition, since fluorescent images excited completely in time with light of a plurality of different or two wavelengths can be acquired, fast-changing phenomena and fast-moving samples can be observed.
In addition, since an optimum excitation filter can be selected according to the reflection peak wavelength of the mirror, it is not necessary to use an excitation filter having a characteristic with a plurality of peak wavelengths such as an expensive dual peak excitation filter, resulting in cost reduction. Can be reduced.

With the configuration as in this onset bright, to take the excitation light and fluorescence another optical path, dichroic mirrors and semi-transparent mirror is not necessary to separate the exciting light and fluorescence put. For this reason, excitation light and fluorescence can be transmitted efficiently, which is particularly effective when a dark fluorescent sample is observed.
Further, since it is not necessary to use an expensive dual dichroic mirror, the cost can be reduced.

Further, the microscopic image processing apparatus according to the onset Akira includes an irradiation light quantity adjusting means to adjust the intensity of the irradiation morphism light.

This onset Ming, since the such a configuration, it is possible to alter the balance of a plurality or two excitation light intensity freely, the intensity of fluorescence corresponding to one of the excitation light corresponding to the other excitation light Even if it is extremely stronger than the intensity of fluorescence, the dynamic range of the camera can be used effectively by adjusting the balance of excitation light intensity and making the fluorescence intensity corresponding to both excitation lights equal. .
In addition, it is not necessary to switch the neutral density filter in time, the generation of unnecessary vibration is suppressed, and the focal position due to vibration is not shifted.

Further, the microscope image processing apparatus according to the onset Akira includes an irradiation light polarization direction selecting means for selecting the polarization direction of the illumination light of multiple.

With the configuration as in this onset bright, the excitation spectrum and a bimodal, the form of the excitation spectrum, for example, when a fluorescent substance is used that varies depending on the concentration of calcium ions, polarization directions are perpendicular to each other The sample is excited simultaneously with excitation light of two wavelengths to generate fluorescence. Among the fluorescence, by separating and imaging components having the same polarization direction as each excitation light, and by calculating the ratio of the two obtained images and measuring the ratio image, For example, the change in the concentration of calcium ions can be measured at the same time without causing any time lag, and it is possible to measure even a fast-changing phenomenon or a fast-moving sample.

Further, the microscope image processing apparatus according to the onset bright preferably comprises a wavelength distribution monitoring means for monitoring the wavelength distribution of the light irradiated multiple.

With the configuration as this, it is possible to reliably monitor the wavelength distribution of light incident on the sample.

Further, the microscope image processing apparatus according to the onset Ming, mirror is preferably a semi-transparent mirror.

With the configuration as this, the wavelength dependency on the reflectivity and the transmittance hardly By using semi Torukyo, without being constrained to the reflection characteristics of the mirror, the excitation filter and fluorescence filter in accordance with the fluorescent substance to be used optimally You can choose. Further, since it is not necessary to use an expensive dual dichroic mirror, the cost can be reduced.

Further, the microscope image processing apparatus according to the onset Ming is preferably irradiated light beam splitting means and the irradiation light beam combining means is a dichroic mirror.

With the configuration as this minimizes the loss of the excitation light in comparison with the case of using semi-transparent mirror as the irradiation light beam splitting means and the irradiation light beam combining means, it is possible to excite the sample, in particular a dark sample It is effective when observing.

Further, the microscope image processing apparatus according to the onset Ming is preferably irradiated light beam splitting means and the irradiation light beam combining means is a polarizing beam splitter.

As this, the use of polarization beam splitter as the irradiation light beam splitting means and the irradiation light beam combining means, as possible out to simplify the optical system. Further, there is less loss of light emitted from the light source than using a semi-transparent mirror, and the sample can be efficiently illuminated with light.

Incidentally, the microscopic image processing apparatus according to the onset Ming, at least one of the wavelength selection means double the number, not the preferred being disposed to be inserted into and removed from the split light path by the optical path splitting means.

  Hereinafter, each embodiment will be described with reference to the drawings.

FIG. 1 is a schematic configuration diagram of an illumination apparatus for a fluorescence microscope and an image processing apparatus using the illumination apparatus according to Embodiment 1 of the present invention.
The fluorescent microscope illumination apparatus of the present embodiment and the image processing apparatus using the illumination apparatus include a white light source 11 that is turned on by an arc, a filament, an LED, or the like, and a light beam emitted from the light source 11 as a first irradiation light. A semi-transparent mirror 21 that is a light beam splitting unit that branches into a light beam and a light beam of the second irradiation light, an excitation filter 24A that is a first wavelength selection unit that selects the wavelength of the first irradiation light, and a second irradiation The excitation filter 24B, which is the second wavelength selection means for selecting the wavelength of the light, and the light beam of the first irradiation light whose wavelength is selected and the light beam of the second irradiation light whose wavelength is selected are a single light beam. A semi-transmission mirror 25 which is a light beam synthesis means to synthesize the light beam, a dichroic mirror 41 which is a mirror that transmits the light beam synthesized by the semi-transmission mirror 25 to the sample 43 and transmits the light from the sample 43, Dichroic An objective lens 42 disposed between the mirror 41 and the sample 43, and an imaging device that separates the fluorescence from the sample 43 that has passed through the objective lens 42 and the dichroic mirror 41 into fluorescence excited at the respective wavelengths and captures an image. A certain camera 53A, 53B and an image processing unit 61, which is an image processing means for processing a fluorescent image captured by the camera 53A, 53B, are configured.

  The light source 11 is composed of a light source using a mercury lamp or a xenon lamp that emits light in the ultraviolet wavelength region to the visible wavelength region. The light emitted from the light source 11 enters the collector lens 12. The collector lens 12 is configured to convert light from the light source 11 into a parallel light beam. The light converted into the parallel light beam through the collector lens 12 is incident on the semi-transparent mirror 21 which is a light beam splitting means.

The semi-transparent mirror 21 has a function of reflecting a part of the incident light beam and transmitting the rest.
A reflecting mirror 22A, a neutral density filter 23A, and an excitation filter 24A are arranged on the optical path of the light beam A that has passed through the semi-transparent mirror 21. A reflecting mirror 22B, a neutral density filter 23B, and an excitation filter 24B are arranged on the optical path of the light beam B reflected by the semi-transparent mirror 21.

The reflecting mirror 22A and the reflecting mirror 22B are provided with an inclination adjusting mechanism (not shown) for completely matching the traveling direction and the position on the optical path after the light beams A and B pass through the semi-transparent mirror 25.
The neutral density filters 23A and 23B can individually and independently adjust the light amount of the light beam A and the light amount of the light beam B, respectively.
Further, the neutral density filters 23A and 23B are provided so as to be easily inserted into and removed from the optical paths of the luminous flux A and the luminous flux B through turrets or sliders, respectively.
The excitation filters 24A and 24B have a property of transmitting only light in a specific wavelength region in the light beams A and B, respectively, and can be easily inserted into and removed from the optical paths of the light beams A and B through turrets or sliders, respectively. It is provided as possible.

  The semi-transparent mirror 25 has a characteristic of transmitting a part of the light beam A and reflecting a part of the light beam B. The light flux A and the light flux B after passing through the semi-transparent mirror 25 are completely matched in the traveling direction and the position on the optical path by adjusting the tilt of the reflecting mirror 22A and the reflecting mirror 22B through the tilt adjusting mechanism described above. ing.

In the figure, 31 is a projection lens that guides the light source images of the luminous flux A and the luminous flux B synthesized by the semi-transparent mirror 25 to the pupil plane of the objective lens 42, and 44 is a reflection mirror 45 that emits fluorescence from the sample 43 that has passed through the dichroic mirror 41. This is an imaging lens that reflects and forms an image on the imaging surfaces of the cameras 53A and 53B.
The dichroic mirror 41 is configured using a dual dichroic mirror having reflection characteristics having two reflection peak wavelengths. The dichroic mirror 41 reflects light transmitted through the projection lens 31 toward the objective lens 42 and is emitted from the sample 43. It is designed to transmit fluorescence.

In the figure, 51 is a dichroic mirror that transmits or reflects the fluorescence from the sample 43 depending on its wavelength, and 52A and 52B are specific light beams of a light beam A ′ and a light beam B ′ divided through the dichroic mirror 51, respectively. It is a fluorescent filter that transmits only the wavelength region.
The fluorescent filters 52A and 52B are provided so as to be easily inserted into and removed from the optical paths of the light beams A ′ and B ′ via turrets or sliders, respectively.
The cameras 53A and 53B are provided so as to capture the light flux A ′ and the light flux B ′ that have passed through the fluorescent filters 52A and 52B.

The image processing unit 61 is configured to accumulate electrical signals output from the camera 53A and the camera 53B in a memory and to perform various calculations on the obtained fluorescence image of the sample 43.
In the figure, 62 is an image display unit. The image display unit 62 has a function of displaying the image processed by the image processing unit 61.

  In this embodiment, a semi-transmissive mirror 71 is disposed between the semi-transmissive mirror 25 and the projection lens 31, and a part of the light beams A and B reflected and transmitted by the semi-transmissive mirror 25 are half-reflected. The light is incident on the spectroscope 72 through the transmission mirror 71. The spectroscope 72 has a function of measuring the wavelength distribution of each of the incident light beams A and B.

According to the fluorescence microscope illumination apparatus and the image processing apparatus using the illumination apparatus of the present embodiment configured as described above, when the light source 11 is turned on, the light emitted from the light source 11 passes through the collector lens 12. It is converted into a parallel light beam and is split into two light beams A and B via the semi-transparent mirror 21.
The light beam A transmitted through the semi-transparent mirror 21 is reflected by the reflecting mirror 22A, and then passes through the neutral density filter 23A with a predetermined transmittance. Further, light in a predetermined wavelength region is transmitted through the excitation filter 24 </ b> A and is incident on the semi-transparent mirror 25. The light beam A that has been transmitted through the semi-transparent mirror 25 and transmitted through the semi-transparent mirror 71 passes through the projection lens 31, is reflected by the dichroic mirror 41, and irradiates the sample 43 through the objective lens 42. The sample 43 is excited by being irradiated with the light beam A and emits fluorescence.
The fluorescence emitted from the sample 43 by irradiating the light beam A travels backward through the objective lens 42, passes through the dichroic mirror 41 and the imaging lens 44, is reflected by the reflecting mirror 45, and is reflected by the dichroic mirror 51. Next, the light passes through the fluorescent filter 52A and is captured as a fluorescent image by the camera 53A.

On the other hand, the light beam B reflected by the semi-transparent mirror 21 is reflected by the reflecting mirror 22B and then passes through the neutral density filter 23B with a predetermined transmittance. Further, the light passes through the excitation filter 24 </ b> B in a predetermined wavelength region and enters the semi-transparent mirror 25. The light beam B reflected from the semi-transparent mirror 25 and transmitted through the semi-transparent mirror 71 passes through the projection lens 31, is reflected by the dichroic mirror 41, and irradiates the sample 43 through the objective lens 42. The sample 43 is excited by being irradiated with the light beam B and emits fluorescence.
The fluorescence emitted from the sample 43 by irradiating the light beam B travels backward through the objective lens 42, passes through the dichroic mirror 41 and the imaging lens 44, is reflected by the reflecting mirror 45, and passes through the dichroic mirror 51. Next, the light passes through the fluorescent filter 52B and is captured as a fluorescent image by the camera 53B.

Further, the fluorescence image of the sample 43 captured by the cameras 53A and 53B is calculated by the image processing unit 61, and the luminance ratio between the images is calculated, and the original image and the ratio image are displayed on the image display unit 62.
Further, part of the light beams A and B that have passed through the semi-transparent mirror 25 are reflected by the semi-transparent mirror 71 and incident on the spectroscope 72, and their respective wavelength distributions are monitored.

Therefore, according to the illumination device of the fluorescence microscope and the image processing device using the illumination device of the present embodiment, the sample can be illuminated in two different wavelength ranges with only one light source 11, so that the conventional plural Thus, uniform illumination is possible without being affected by differences in spatial intensity distribution such as unevenness in the field of view of each light source.
In addition, since the sample can be excited in two different wavelength ranges by using only one light source 11, the characteristic change accompanying the time-dependent change of each light source, such as a conventional apparatus using a plurality of light sources, A ratio image can be reliably obtained without being affected by differences in temporal fluctuations such as noise.
In addition, since fluorescence images excited at two different wavelengths can be acquired at the same time in time, a fast-changing phenomenon and a fast-moving sample can be observed with multiple fluorescence.
Further, since an optimum excitation filter can be selected according to the reflection peak wavelength of the dichroic mirror 41, it is not necessary to use an expensive dual peak excitation filter, and as a result, the cost can be reduced.
Furthermore, since the balance between the two excitation light intensities can be freely changed, even if the fluorescence intensity corresponding to one excitation light is extremely stronger than the fluorescence intensity corresponding to the other excitation light, the excitation light By adjusting the intensity balance and making the fluorescence intensities corresponding to both excitation lights equal, the dynamic range of the camera can be used effectively.
In addition, it is not necessary to switch the neutral density filter in time, generation of unnecessary vibration can be suppressed, and the focal position due to vibration does not shift.
Further, the wavelength distribution of the light incident on the microscope can be reliably monitored by the spectroscope 72.

FIG. 2 is a schematic configuration diagram illustrating a modification of the illumination unit of the fluorescence microscope according to the modification of the first embodiment of the present invention. The same components as those in the embodiment of FIG. 1 are denoted by the same reference numerals, and detailed description thereof is omitted.
In the configuration shown in FIG. 1, the semi-transparent mirrors 21 and 25 are used for the light beam splitting means and the light beam combining means, respectively, but instead of them, dichroic mirrors 28A and 28B may be used as shown in FIG. The dichroic mirrors 28A and 28B have characteristics of reflecting incident light having a shorter wavelength than a certain wavelength λ and transmitting incident light having a longer wavelength than the wavelength λ.
In this modification, an excitation filter having a transmission wavelength range longer than the wavelength λ is used as the excitation filter 24A, and an excitation filter having a transmission wavelength range shorter than the wavelength λ is used as the excitation filter 24B.

According to the illumination unit of the present modification configured as described above, the light having a wavelength longer than the wavelength λ from the light source 11 is transmitted through the dichroic mirror 28A, reflected by the reflecting mirror 22A, and the neutral density filter 23A. Then, the light passes through the excitation filter 24A, passes through the dichroic mirror 28B, and is guided to the sample 43 side. On the other hand, light having a wavelength shorter than the wavelength λ from the light source 11 is reflected by the dichroic mirror 28A, reflected by the reflecting mirror 22B, transmitted through the optical filter 23B and the excitation filter 24B, and reflected by the dichroic mirror 28B. Guided to the sample 43 side.
Therefore, according to the light source unit of the present modification, the loss of excitation light is minimized as compared with the case where the half mirrors 21 and 25 as shown in FIG. Since the sample can be excited, it is particularly effective when observing a dark sample.

The semi-transparent mirrors 21 and 25 or the dichroic mirrors 28A and 28B in the first embodiment are merely examples of light beam dividing means or light beam synthesizing means, and are not limited thereto. As another example of the beam splitting unit or the beam combining unit, for example, a two-branch bundle fiber (not shown) may be used.
When a bifurcated bundle fiber is used instead of the semi-transparent mirror 21, the combined end of the bundle fiber may be disposed immediately after the collector lens 12, and the branched end of the bundle fiber may be disposed immediately before the reflecting mirrors 22A and 22B. .
In this case, the light emitted from the light source 11 can be incident on the reflecting mirrors 22A and 22B if it can be incident on the bundle fiber without being made into a parallel light beam by the collector lens 12. The system can be easily adjusted.

Further, when a bifurcated bundle fiber is used in place of the semi-transparent mirror 25, the branch ends of the bundle fiber may be arranged immediately after the excitation filters 24A and 24B, respectively.
In this way, since the light transmitted through the excitation filters 24A and 24B can be combined so as to be incident on each of the branch ends of the bundle fiber, the neutralization filters 23A and 23B and the excitation filters 24A and 24B can be freely arranged. The degree can be increased.

  The excitation filters 24A and 24B in the first embodiment are merely examples of wavelength selection means, and are not limited to these. As another example of the wavelength selection unit, for example, a monochromator may be used instead of the excitation filters 24A and 24B. In this way, it is not necessary to prepare a plurality of excitation filters in advance.

  A semi-transparent mirror may be used instead of the dichroic mirror 51. In this case, since the wavelength characteristics of the reflectance and transmittance of the semi-transparent mirror are almost uniform, each time the fluorescent material in the sample is changed, as in the case of using the dichroic mirror 51, transmission or reflection is performed according to the fluorescence. Therefore, it is possible to save the trouble of having to replace the dichroic mirror 51 with the characteristic to be changed.

FIG. 3 is a schematic configuration diagram of a fluorescent microscope illumination device and an image processing apparatus using the illumination device according to a second embodiment of the present invention. The same components as those in the embodiment of FIG. 1 are denoted by the same reference numerals, and detailed description thereof is omitted.
In the second embodiment, a polarizing plate 26A is disposed on the optical path of the light beam A transmitted through the semi-transparent mirror 21 in addition to the reflecting mirror 22A, the neutral density filter 23A, and the excitation filter 24A. In addition to the reflecting mirror 22B, the neutral density filter 23B, and the excitation filter 24B, a polarizing plate 26B is disposed on the optical path of the light beam B reflected by the semi-transparent mirror 21. The polarizing plates 26A and 26B have a characteristic of selecting the polarization direction of the incident light beam from the light source 11.

In the second embodiment, the fluorescent filter 52 and the semi-transparent mirror 56 that divides the light beam transmitted through the fluorescent filter 52 into the light beam A ′ and the light beam B ′ are arranged on the optical path on the reflection side of the reflecting mirror 45. A polarizing plate 54A and a polarizing plate 54B are arranged on the optical path of the two light beams A ′ and B ′ divided through 56, and the fluorescence transmitted through the polarizing plates 54A and 54B is captured by the cameras 53A and 53B, respectively. It has become so.
The fluorescent filter 52 has a characteristic of transmitting only light in a specific wavelength region out of light from the sample 43.
The polarizing plates 54A and 54B have a characteristic of transmitting only light of a specific polarization direction component through the light beams A ′ and B ′ divided by the semi-transparent mirror 56, respectively.

Here, the directional relationship of the transmission polarization axes of the polarizing plate 26A and the polarizing plate 26B, and the polarizing plates 54A and 54B will be described.
The polarizing plate 26A and the polarizing plate 26B have a transmission polarization axis perpendicular to each other, and the polarizing plate 54A and the polarizing plate 54B have a transmission polarization axis perpendicular to each other. Further, the polarizing plate 26A and the polarizing plate 54A have transmission polarization axes parallel to each other, and the polarizing plate 26B and the polarizing plate 54B have transmission polarization axes parallel to each other.
In this way, the component of the fluorescence of the sample 43 excited by the light beam A having a predetermined polarization direction that has passed through the polarizing plate 26A is dominant in the component parallel to the polarization direction of the excitation light (light beam A). Therefore, although the component can permeate | transmit the polarizing plate 54A, it cannot permeate | transmit the polarizing plate 54B. On the other hand, the component perpendicular to the polarization direction of the excitation light (light flux A) in the fluorescence of the sample 43 can pass through the polarizing plate 54B, but cannot pass through the polarizing plate 54A. On the other hand, the component parallel to the polarization direction of the excitation light (light beam B) is dominant in the polarization direction of the fluorescence of the sample 43 excited by the light beam B having a predetermined polarization direction transmitted through the polarizing plate 26B. The component can pass through the polarizing plate 54B, but cannot pass through the polarizing plate 54A. Conversely, the component perpendicular to the polarization direction of the excitation light (light beam B) in the fluorescence of the sample 43 can pass through the polarizing plate 54A, but cannot pass through the polarizing plate 54B.

According to the fluorescence microscope illumination apparatus and the image processing apparatus using the illumination apparatus of the present embodiment configured as described above, when the light source 11 is turned on, the light emitted from the light source 11 passes through the collector lens 12. It is converted into a parallel light beam, enters the semi-transparent mirror 21, and is divided into two light beams A and B.
The light beam A transmitted through the semi-transparent mirror 21 is reflected by the reflecting mirror 22A, and then passes through the neutral density filter 23A with a predetermined transmittance. In addition, light in a predetermined wavelength band is transmitted through the excitation filter 24A, and light having a predetermined polarization direction is selected via the polarizing plate 25A and is incident on the semi-transparent mirror 25. The light beam A that has been transmitted through the semi-transparent mirror 25 and transmitted through the semi-transparent mirror 71 passes through the projection lens 31, is reflected by the dichroic mirror 41, and irradiates the sample 43 through the objective lens 42. The sample 43 is excited by being irradiated with the light beam A and emits fluorescence.
In this embodiment, a fluorescent substance having a large molecular weight such as GFP (green fluorescent protein) or RFP (red fluorescent protein) is used as the fluorescent substance in the sample 43. When the molecular weight is large, the rotational movement of the fluorescent material is slow, and the polarization direction of the fluorescence emitted from the sample 43 is dominant in the component parallel to the polarization direction of the excitation light irradiated on the sample, and the parallel component and The ratio of the component perpendicular to the polarization direction of the excitation light is substantially constant.
The fluorescence emitted from the sample 43 by irradiating the light beam A travels backward through the objective lens 42, passes through the dichroic mirror 41 and the imaging lens 44, reflects off the reflecting mirror 45, and passes through the fluorescent filter 52, It is transmitted and reflected by the semi-transparent mirror 56. Next, a component of the fluorescence parallel to the polarization direction of the light flux A is transmitted through the polarizing plate 54A and captured as a fluorescent image by the camera 53A, and a vertical component is transmitted through the polarizing plate 54B and is captured as a fluorescent image by the camera 53B. Imaged.

On the other hand, the light beam B reflected by the semi-transparent mirror 21 is reflected by the reflecting mirror 22B and then transmitted by the neutral density filter 23B with a predetermined transmittance. In addition, light in a predetermined wavelength region is transmitted through the excitation filter 24B, and light having a predetermined polarization direction is selected via the polarizing plate 25B and is incident on the semi-transparent mirror 25. The light beam B reflected by the semi-transparent mirror 25 and transmitted through the semi-transparent mirror 71 passes through the projection lens 31, is reflected by the dichroic mirror 41, and irradiates the sample 43 through the objective lens 42. The sample 43 is excited by being irradiated with the light beam B and emits fluorescence.
The fluorescence emitted from the sample 43 by irradiating the light beam B travels backward through the objective lens 42, passes through the dichroic mirror 41 and the imaging lens 44, reflects off the reflecting mirror 45, and passes through the fluorescent filter 52, The semi-transparent mirror 56 is transmitted and reflected. Next, a component of the fluorescence parallel to the polarization direction of the light beam B is transmitted through the polarizing plate 54B and captured as a fluorescent image by the camera 53B, and a vertical component is transmitted through the polarizing plate 54A and as a fluorescent image by the camera 53A. Imaged.

Next, effects of the configuration and operation of the present embodiment will be described.
In the case of using a fluorescent material whose excitation spectrum is bimodal, such as GFP, and the shape of the excitation spectrum changes depending on the concentration of calcium ions, for example, conventionally, there are two peaks corresponding to the bimodal. It has been difficult to extract changes in the concentration of calcium ions from fluorescence emitted by simultaneously applying excitation light of a wavelength.
However, according to the illumination apparatus of the fluorescence microscope and the image processing apparatus using the illumination apparatus of the present embodiment, as described above, the sample is simultaneously excited by the excitation light having two wavelengths whose polarization directions are perpendicular to each other, and fluorescence is emitted. After the two components whose polarization directions are orthogonal to each other are imaged simultaneously with two cameras, and calculated based on the two images obtained, they are excited independently with the excitation light of each wavelength. In this case, a fluorescent image equivalent to the fluorescent image obtained can be obtained separately. Furthermore, by measuring the ratio image by calculating the ratio of the two obtained images, for example, it is possible to measure changes in the concentration of calcium ions at exactly the same time without causing any time lag. It is possible to measure even a fast-changing phenomenon or a fast-moving sample.

In the second embodiment, the polarizing plates 26A and 26B are used as the polarization direction selecting means, but a polarizing beam splitter may be used instead.
FIG. 4 is a schematic configuration diagram of a fluorescent microscope illumination apparatus and an image processing apparatus using the illumination apparatus according to a modification of the second embodiment of the present invention. The same parts as those in FIG. 1 and FIG.
In this modified example, polarization beam splitters 27A and 27B are used as shown in FIG. 4 instead of the semi-transparent mirrors 21 and 25 shown in FIG.
The polarization beam splitter 27A is disposed at a position where the parallel light beam that has passed through the collector lens 12 enters. The polarization beam splitter 27A has a characteristic of transmitting light having a polarization direction component parallel to the paper surface of the incident light beam and reflecting light having a polarization direction component perpendicular to the paper surface. The light beam is divided into light beams A and B according to the polarization direction.
A reflecting mirror 22A, a neutral density filter 23A, and an excitation filter 24A are arranged on the optical path of the light beam A that has passed through the polarization beam splitter 27A. A reflecting mirror 22B, a neutral density filter 23B, and an excitation filter 24B are arranged on the optical path of the light beam B reflected by the polarization beam splitter 27A.

The polarization beam splitter 27B is disposed at a position where the light beam A having passed through the excitation filter 24A and the light beam B having passed through the excitation filter 24B are incident. The polarization beam splitter 27B has a function similar to that of the polarization beam splitter 27A, transmits a light beam A having a polarization component parallel to the paper surface, and has a component in a polarization direction perpendicular to the paper surface. Is reflected to synthesize the light flux A and the light flux B.
In the present modification, a fluorescent filter 52 is provided on the optical path on the reflection side of the reflecting mirror 45, and a polarizing beam splitter 55 that splits the light beam transmitted through the fluorescent filter into a light beam A 'and a light beam B' according to the difference in polarization direction. The fluorescent light arranged and divided by the polarization beam splitter 55 is imaged by the cameras 53A and 53B, respectively.
The polarization beam splitter 55 has a characteristic of transmitting light having a polarization direction component parallel to the paper surface of the incident light beam and reflecting light having a polarization direction component perpendicular to the paper surface. It is configured to be divided into light beams A ′ and B ′ according to the polarization direction.

According to the illumination apparatus of the fluorescence microscope of the present modification and the image processing apparatus using the illumination apparatus configured as described above, the polarization beam splitters 27A and 27B include the beam splitting unit in addition to the function of the polarization direction selection unit. In addition, since it also has the function of the light beam combining means, the semi-transparent mirror 21 and the semi-transparent mirror 25 can be omitted, and the optical system can be simplified. Further, in this modification, when the polarizing beam splitters 27A and 27B are used, the loss of light from the light source 11 is reduced as compared with the case where the semi-transparent mirrors 21 and 25 are used. Can do. Further, since the polarizing beam splitter 55 plays the role of the semi-transparent mirror 51 and the polarizing plates 54A and 54B in FIG. 3, not only the optical system can be simplified but also the stability of the system can be improved. In addition, when the polarization beam splitter 55 is used, the loss of light emitted from the sample is reduced as compared with the case where the semi-transparent mirror 56 is used, so that fluorescence can be detected efficiently.
In addition, according to the present modification, the sample can be excited in two different wavelength ranges with only one light source 11, and thus the same effect as in the first embodiment shown in FIGS. Moreover, it has the same effect as the structure shown in FIG.

FIG. 5 is a schematic configuration diagram of an illumination apparatus for a fluorescence microscope and an image processing apparatus using the illumination apparatus according to Example 3 of the present invention. The same components as those in the embodiment of FIG. 1 are denoted by the same reference numerals, and detailed description thereof is omitted.
In the third embodiment, a semi-transparent mirror 46 is arranged in place of the dichroic mirror 41 in the apparatus of the first embodiment shown in FIG. The semi-transparent mirror 46 has a function of reflecting a part of incident light and transmitting a part thereof, and has almost no wavelength dependency of the reflectance and transmittance. A part of the luminous flux A and the luminous flux B synthesized by the semi-transparent mirror 25 is reflected to the objective lens 42 side, and a part of the fluorescence emitted from the sample 43 is transmitted.

According to the fluorescent microscope illumination apparatus and the image processing apparatus using the illumination apparatus of the present embodiment configured as described above, since the semi-transparent mirror 46 having almost no wavelength dependency in reflectance and transmittance is used, the dichroic is used. Unlike the embodiment using the mirror 41, the reflection characteristics are not limited, and the excitation filter and the fluorescence filter can be optimally selected according to the fluorescent material used for the sample 43. Further, since it is not necessary to use an expensive dual dichroic mirror, the cost can be reduced.
In addition, since the sample can be excited in two different wavelength ranges with only one light source 11, the same effects as those of the first embodiment can be obtained.

FIG. 6 is a schematic configuration diagram of a fluorescent microscope illumination device and an image processing apparatus using the illumination device according to a fourth embodiment of the present invention. The same components as those in the embodiment of FIG. 1 are denoted by the same reference numerals, and detailed description thereof is omitted.
The fourth embodiment is different from the first embodiment shown in FIG. 1 in the configuration on the optical path from the semi-transparent mirror 71 to the reflecting mirror 45. On the optical path between these, the projection lens 31, the reflecting mirror 47, and The objective lens 42A, which is the first objective lens, the objective lens 42B, which is the second objective lens, and the imaging lens 44 are arranged so as to perform transmission-type fluorescence observation.
The projection lens 31 guides the light source images of the light beams A and B synthesized by the semi-transparent mirror 25 to the pupil plane of the first objective lens 42A. The reflecting mirror 47 reflects the light transmitted through the projection lens 31 and guides it to the objective lens 42A. The objective lens 42 </ b> A is disposed so as to irradiate the sample 43 with the light beam synthesized by the semi-transparent mirror 25. The objective lens 42B is disposed so as to face the objective lens 42A with the sample 43 interposed therebetween. The objective lens 42B transmits the fluorescence emitted from the sample 43 and reflects it with the reflecting mirror 45 via the imaging lens 44, whereby the camera 53A and An image is formed on the image pickup surface 53B. Further, the first objective lens 42A can move up and down along the optical axis direction to adjust the diameter of the light applied to the sample 43.

According to the fluorescence microscope illumination apparatus and the image processing apparatus using the illumination apparatus of the present embodiment configured as described above, the excitation light and the fluorescence follow different optical paths, so that the dichroic mirror 41 shown in FIG. Or it is not necessary to separate excitation light and fluorescence using the semi-transparent mirror 46 shown in FIG. For this reason, excitation light and fluorescence can be transmitted efficiently, which is particularly effective when observing a dark fluorescent sample. Further, since it is not necessary to use an expensive dual dichroic mirror, the cost can be reduced.
In addition, since the sample can be excited in two different wavelength ranges with only one light source 11, the same effect as in the first embodiment can be obtained.
Also in the fourth embodiment, it is of course possible to configure the illumination unit of the microscope as shown in FIG. 2, and in that case, the same effect can be obtained.

FIG. 7 is a schematic configuration diagram of a fluorescent microscope illumination device and an image processing apparatus using the illumination device according to a fifth embodiment of the present invention. The same components as those in the embodiment of FIG. 3 are denoted by the same reference numerals, and detailed description thereof is omitted.
The fifth embodiment is different from the second embodiment shown in FIG. 3 in the configuration on the optical path from the semi-transparent mirror 25 and the semi-transparent mirror 71 to the reflecting mirror 45, and on the optical path between them, as in the fourth embodiment, A projection lens 31, a reflecting mirror 47, an objective lens 42A that is a first objective lens, an objective lens 42B that is a second objective lens, and an imaging lens 44 are arranged.

According to the illumination apparatus of the fluorescence microscope of the present embodiment and the image processing apparatus using the illumination apparatus configured as described above, the excitation light and the fluorescence follow different optical paths as in the fourth embodiment. It is no longer necessary to separate excitation light and fluorescence using the dichroic mirror 41 or semi-transparent mirror shown in FIG. For this reason, excitation light and fluorescence can be transmitted efficiently, which is particularly effective when a dark fluorescent sample is observed. Further, since it is not necessary to use an expensive dual dichroic mirror, the cost can be reduced.
Other effects are almost the same as those of the second embodiment shown in FIG.

FIG. 8 is a schematic configuration diagram of a fluorescent microscope illumination device and an image processing apparatus using the illumination device according to a sixth embodiment of the present invention. The same components as those in the embodiment of FIG. 4 are denoted by the same reference numerals, and detailed description thereof is omitted.
In the sixth embodiment, the configuration on the optical path from the polarizing beam splitter 27B and the semi-transmissive mirror 71 to the reflecting mirror 45 is different from the modification of the second embodiment shown in FIG. A reflecting mirror 47, an objective lens 42A that is a first objective lens, an objective lens 42B that is a second objective lens, and an imaging lens 44 are disposed.

According to the illumination apparatus of the fluorescence microscope of the present embodiment and the image processing apparatus using the illumination apparatus configured as described above, the excitation light and the fluorescence follow different optical paths as in the fourth embodiment. There is no need to separate excitation light and fluorescence using the dichroic mirror 41 or semi-transparent mirror shown in FIG. For this reason, excitation light and fluorescence can be transmitted efficiently, which is particularly effective when observing a dark fluorescent sample. Further, since it is not necessary to use an expensive dual dichroic mirror, the cost can be reduced.
Other effects are almost the same as those of the modification of the second embodiment shown in FIG.

FIG. 9 is a schematic configuration diagram of a fluorescent microscope illumination device and an image processing apparatus using the illumination device according to a seventh embodiment of the present invention. The same components as those in the embodiment of FIG. 1 are denoted by the same reference numerals, and detailed description thereof is omitted.
In the seventh embodiment, the light beam emitted from the light source 11 is branched into three light beams of irradiation light through the semi-transparent mirror 21 and the semi-transparent mirror 81, and is branched by these light beam dividing means through the excitation filters 24A to 24C. In addition, the wavelength of each irradiation light is selected, and the light beams of the plurality of irradiation lights whose wavelengths are selected are combined into a single light beam through the half mirror 82 and the half mirror 25.

The light converted into the parallel light flux through the collector lens 12 enters the semi-transparent mirror 21 which is a light beam splitting means. The semi-transparent mirror 21 has a characteristic of reflecting a part of the incident light beam and transmitting the rest.
On the optical path of the light beam A that has passed through the semi-transparent mirror 21, a semi-transparent mirror 81 is disposed as a light beam dividing unit. The semi-transparent mirror 81 has a characteristic of reflecting a part of the incident light beam and transmitting the rest.
A reflecting mirror 22A, a neutral density filter 23A, and an excitation filter 24A are arranged on the optical path of the light beam A that has passed through the semi-transparent mirror 81.
Further, a neutral density filter 23C, an excitation filter 24C, and a semi-transparent mirror 82 are arranged on the optical path of the light beam C reflected by the semi-transparent mirror 81.
On the other hand, a neutral density filter 23B, an excitation filter 24B, and a reflecting mirror 22B are disposed on the optical path of the light beam B reflected by the semi-transparent mirror 21.

The reflecting mirrors 22A and 22B and the semi-transparent mirrors 81 and 82 include an inclination adjusting mechanism (not shown) for completely matching the traveling direction of the light beam A, the light beam B, and the light beam C after passing through the semi-transparent mirror 25 and the position on the optical path. ) Is provided.
The neutral density filters 23A, 23B, and 23C can individually adjust the light amount of the light beam A, the light amount of the light beam B, and the light amount of the light beam C, respectively.
The neutral density filters 23A, 23B, and 23C are provided so as to be easily inserted into and removed from the optical paths of the light beams A, B, and C through turrets or sliders, respectively.
The excitation filters 24A, 24B, and 24C have characteristics of transmitting only light in a specific wavelength region in the light flux A, the light flux B, and the light flux C, respectively, and the light flux A and the light flux B through a turret or a slider, respectively. , And the light path of the light beam B so as to be easily inserted and removed.

  The semi-transparent mirror 82 has a characteristic of transmitting a part of incident light and reflecting a part of the incident light. The parallel light beam B transmitted through the semi-transparent mirror 82 and the parallel light beam C reflected from the semi-transparent mirror are combined to produce a semi-transparent mirror. 25. Further, the semi-transparent mirror 25 has a characteristic of transmitting a part of incident light and reflecting a part thereof, and the parallel light beam A transmitted through the semi-transparent mirror 25 and the parallel light beams B and C reflected from the semi-transparent mirror 25 are combined. It has come to be. At that time, by adjusting the inclination of the reflecting mirror 22A, the reflecting mirror 22B, and the semi-transparent mirrors 81 and 82 through the above-described inclination adjusting mechanism, the parallel light fluxes A, B, and C that have passed through the semi-transparent mirror 25 are traveling directions. And the position on the optical path is completely matched.

In this embodiment, the dichroic mirror 41 is a multi-chromic mirror having reflection characteristics having three or more reflection peaks. The excitation filters 24A, 24B, and 24C are selected according to the reflection peak of the dichroic mirror 41.
In this embodiment, as the camera 91, a three-plate type color CCD camera that divides the fluorescence from the sample 43 into three wavelength components and takes an image is used. The dichroic as shown in the embodiment of FIG. Optical members corresponding to the mirror 51 and the fluorescent filters 52A and 52B are omitted.

According to the fluorescence microscope illumination apparatus and the image processing apparatus using the illumination apparatus of the present embodiment configured as described above, the sample can be excited in three different wavelength ranges with only one light source 11. The same effects as those of the first embodiment are obtained. Furthermore, it is possible to simultaneously excite a sample in three or more wavelength ranges and observe three or more fluorescences simultaneously. For example, in addition to the change in calcium ion concentration, simultaneously observe the change in chloride ion concentration. The effect that can be done.
In addition, although the example which divides | segments an irradiation light beam into three was shown in a present Example, four or more division | segmentation is also possible. Further, if a semi-transparent mirror is used instead of the dichroic mirror 41, the same effect as in the third embodiment can be obtained.

In the example of FIG. 9, a three-plate color CCD camera that divides the fluorescence from the sample 43 into three wavelength components and captures an image is used as the camera 91. However, the dichroic as shown in the embodiment of FIG. A mirror 51 may be used to divide into three optical paths, and a fluorescent filter and a single-plate CCD camera may be arranged in each optical path.
FIG. 10 is a schematic configuration diagram of a fluorescent microscope illumination device and an image processing apparatus using the illumination device according to a modification of the seventh embodiment. The same components as those in the embodiment of FIG. 9 are denoted by the same reference numerals, and detailed description thereof is omitted.
In the present modification, a dichroic mirror 51 is disposed on the optical path on the reflection side of the reflecting mirror 45, and the light beam reflected by the reflecting mirror 45 is divided into a light beam B ′ and other light beams, and further, the dichroic mirror 51. A dichroic mirror 51 ′ is arranged on the optical path of the light beam that has passed through the light beam, and the light beam that has passed through the dichroic mirror 51 is divided into a light beam A ′ and a light beam C ′.
On the optical paths of the divided light beams A ′, B ′, and C ′, fluorescent filters 52A, 52B, and 52C that transmit only a specific wavelength region, and cameras 53A, 53B, and 53C, respectively, are provided. Yes. A single-plate CCD camera is used for each of the cameras 53A, 53B, and 53C.
If configured as in the present modification, the single-plate CCD camera is less expensive than the case of using the three-plate CDD camera shown in FIG. 9, and thus the manufacturing cost of the entire apparatus can be reduced.
In addition, since the fluorescent filter is arranged in each of the optical paths of the light beams A ′, B ′, and C ′ divided into three, even if the fluorescent substance in the sample is changed, the characteristics corresponding to the fluorescence are provided. The fluorescent filter can be inserted into and removed from the optical path of the luminous fluxes A ′, B ′, and C ′, and fluorescence photography can be performed without replacing the CCD camera as in the case of using a three-plate CCD camera.
Other effects are the same as those of the embodiment shown in FIG.

  The configuration of the seventh embodiment shown in FIGS. 9 and 10 relates to the configuration on the optical path from the semi-transparent mirror 25 and the semi-transparent mirror 71 to the reflecting mirror 45, as shown in the embodiment of FIG. The present invention can also be applied to an apparatus having a configuration in which the lens 31, the reflecting mirror 47, the objective lens 42A as the first objective lens, the objective lens 42B as the second objective lens, and the imaging lens 44 are arranged. .

(21) Further, a dichroic mirror is provided between the objective lens and the fluorescent filter, and guides the light beam synthesized by the light beam synthesis means to the sample through the objective lens. The microscope image processing apparatus according to (20) above.

11 Light source 12 Collector lens 21, 25, 56, 46, 71, 81, 82 Semi-transparent mirror 22A, 22B, 45, 47 Reflector 23A, 23B, 23C Attenuating filter 24A, 24B, 24C Excitation filter 26A, 26B, 54A, 54B Polarizing plate 27A, 27B, 55 Polarizing beam splitter 31 Projection lens 28A, 28B, 41, 51, 51 ′ Dichroic mirror 42, 42A, 42B Objective lens 43 Sample 44 Imaging lens 52, 52A, 52B, 52C Fluorescent filter 53A, 53B, 53C Camera 91 Three-plate CCD camera 61 Image processing section 62 Image display section 72 Spectrometer 151 Filter switching means 151a Excitation light selection filter 151b Rotating disk 152a Fluorescence selection filter 158, 164 Dichroic mirror 161A, 16 1B White light source 162A, 162B Collector lens 163A, 163B Excitation filter 165 Observation optical system 171 Irradiation light source 172 Branch optical system 173 Sample

Claims (11)

A white light source,
And irradiating the light beam splitting means for splitting the light flux of the emitted illumination light from the white light source into a plurality of light beams,
An irradiation light wavelength selecting means for selecting a wavelength of the illumination light passing through the optical path respectively provided on the optical path of the split each of the irradiation light,
An irradiation light quantity adjusting means for adjusting the light quantity of the illumination light passing through the optical path respectively provided on the optical path of the split each of the irradiation light,
And irradiating the light beam combining means for light intensity wavelength is selected to synthesize the plurality of the light beam has been adjusted the irradiation light into a single light beam,
A mirror that transmits the fluorescence from the sample excited by該照Shako guides the illumination light made if the direction to be irradiated to the sample,
An objective lens disposed between the sample and the mirror,
A fluorescent light beam splitting means for splitting the fluorescent light beam transmitted through the objective lens and the mirror into a plurality of light beams;
A fluorescence wavelength selection means for selecting the wavelength of the fluorescence that is provided on each of the divided optical paths of the fluorescence and that passes through the optical path;
And an imaging means you capture a fluorescent image by the fluorescent light passing through the optical path provided on an optical path of the fluorescence wavelength is selected,
Image processing means for combining processing a plurality of the fluorescent image is an image shooting,
Image display means for displaying the synthesized fluorescent image;
It has
The irradiation light amount adjusting means can adjust the intensity of the irradiation light by adjusting the light amount of the irradiation light that passes individually,
Microscopic image processing apparatus characterized by the equalize the fluorescence intensity of the fluorescence of each wavelength is selected using at least one of the irradiation light quantity adjusting means and the image processing unit. A white light source,
And irradiating the light beam splitting means for splitting the light flux of irradiation light emitted from the white light source to the light flux of the first light flux of the illumination light and the second illumination light,
A first irradiation light wavelength selection means for selecting a wavelength of the first provided on the optical path of the irradiation light the first irradiation light,
A first irradiation light quantity adjusting means for adjusting the amount of the first provided on the optical path of the irradiation light the first irradiation light,
A second irradiation light wavelength selection means for selecting a wavelength of the second provided on the optical path of the irradiation light the second irradiation light,
A second irradiation light quantity adjusting means for adjusting said second amount of light provided on the optical path of the second irradiation light of the irradiation light,
And irradiating the light beam combining means for combining the light flux of the second irradiation light quantity of light flux and the wavelength of the first irradiation light quantity wavelength is selected is adjusted is selected is adjusted to a single light beam ,
A mirror that transmits the fluorescence from the sample excited by該照Shako guides the illumination light made if the direction to be irradiated to the sample,
An objective lens disposed between the sample and the mirror,
A fluorescent light beam splitting means for splitting the fluorescent light beam transmitted through the objective lens and the mirror into a first fluorescent light beam and a second fluorescent light beam;
First fluorescence wavelength selection means provided on the optical path of the first fluorescence to select the wavelength of the first fluorescence;
Second fluorescence wavelength selection means provided on the optical path of the second fluorescence to select the wavelength of the second fluorescence;
A first imaging means provided on the optical path of the first fluorescence having a selected wavelength, for imaging a first fluorescence image by the first fluorescence;
A second imaging unit that is provided on the optical path of the second fluorescence having a selected wavelength and that captures a second fluorescence image of the second fluorescence;
Image processing means for combining processing and the second fluorescent image and the first fluorescent image,
Image display means for displaying the synthesized fluorescent image;
It has
The first irradiation light intensity adjusting means and the second irradiation light intensity adjusting means can adjust the intensity of the irradiation light by adjusting the intensity of the irradiation light that passes individually. ,
Said first irradiation light quantity adjusting means, and the fluorescence intensity of the second fluorescence and the fluorescence intensity of the first fluorescence by using at least one of the second irradiation light quantity adjusting means and the image processing unit A microscopic image processing apparatus characterized by equalizing. A white light source,
And irradiating the light beam splitting means for splitting the light flux of the emitted illumination light from the white light source into a plurality of light beams,
An irradiation light wavelength selecting means for selecting a wavelength of the illumination light passing through the optical path respectively provided on the optical path of the split each of the irradiation light,
An irradiation light quantity adjusting means for adjusting the light quantity of the illumination light passing through the optical path respectively provided on the optical path of the split each of the irradiation light,
And irradiating the light beam combining means for light intensity wavelength is selected to synthesize the plurality of the light beam has been adjusted the irradiation light into a single light beam,
A first objective lens for guiding the irradiation light which has been made if the direction to be irradiated to the sample,
A second objective lens that fluorescence from the excited sample by the irradiation light combined by being arranged to face the first objective lens across the specimen is incident,
A fluorescent light beam splitting means for splitting the fluorescent light beam incident on the second objective lens into a plurality of light beams;
A fluorescence wavelength selection means for selecting the wavelength of the fluorescence that is provided on each of the divided optical paths of the fluorescence and that passes through the optical path;
And an imaging means you capture a fluorescent image by the fluorescent light passing through the optical path provided on an optical path of the fluorescence wavelength is selected,
Image processing means for combining processing a plurality of the fluorescent image is an image shooting,
Image display means for displaying the synthesized fluorescent image;
It has
The irradiation light amount adjusting means can adjust the intensity of the irradiation light by adjusting the light amount of the irradiation light that passes individually,
Microscopic image processing apparatus characterized by the equalize the fluorescence intensity of the fluorescence of each wavelength is selected using at least one of the irradiation light quantity adjusting means and the image processing unit. A white light source,
And irradiating the light beam splitting means for splitting the light flux of irradiation light emitted from the white light source to the light flux of the first light flux of the illumination light and the second illumination light,
A first irradiation light wavelength selection means for selecting a wavelength of the first provided on the optical path of the irradiation light the first irradiation light,
A first irradiation light quantity adjusting means for adjusting the amount of the first provided on the optical path of the irradiation light the first irradiation light,
A second irradiation light wavelength selection means for selecting a wavelength of the second provided on the optical path of the irradiation light the second irradiation light,
A second irradiation light quantity adjusting means for adjusting said second amount of light provided on the optical path of the second irradiation light of the irradiation light,
And irradiating the light beam combining means for combining the light flux of the second irradiation light quantity of light flux and the wavelength of the first irradiation light quantity wavelength is selected is adjusted is selected is adjusted to a single light beam ,
A first objective lens for guiding the irradiation light which has been made if the direction to be irradiated to the sample,
A second objective lens that fluorescence from the excited sample by the irradiation light combined by being arranged to face the first objective lens across the specimen is incident,
A fluorescent light beam splitting means for splitting the fluorescent light beam incident on the second objective lens into a first fluorescent light beam and a second fluorescent light beam;
First fluorescence wavelength selection means provided on the optical path of the first fluorescence to select the wavelength of the first fluorescence;
Second fluorescence wavelength selection means provided on the optical path of the second fluorescence to select the wavelength of the second fluorescence;
A first imaging means provided on the optical path of the first fluorescence having a selected wavelength, for imaging a first fluorescence image by the first fluorescence;
A second imaging unit that is provided on the optical path of the second fluorescence having a selected wavelength and that captures a second fluorescence image of the second fluorescence;
Image processing means for combining processing and the second fluorescent image and the first fluorescent image,
Image display means for displaying the synthesized fluorescent image;
It has
The first irradiation light intensity adjusting means and the second irradiation light intensity adjusting means can adjust the intensity of the irradiation light by adjusting the intensity of the irradiation light that passes individually. ,
Said first irradiation light quantity adjusting means, and the fluorescence intensity of the second fluorescence and the fluorescence intensity of the first fluorescence by using at least one of the second irradiation light quantity adjusting means and the image processing unit A microscopic image processing apparatus characterized by equalizing. 5. The microscope image processing apparatus according to claim 1, wherein the irradiation light beam splitting unit and the irradiation light beam combining unit are dichroic mirrors. 6. The microscope image processing apparatus according to claim 1, wherein the fluorescent light beam splitting unit is a dichroic mirror. A white light source,
Irradiation light beam splitting means for splitting the light beam of the irradiation light emitted from the white light source into a light beam of the first irradiation light and a light beam of the second irradiation light;
First irradiation light wavelength selection means provided on the optical path of the first irradiation light and selecting the wavelength of the first irradiation light;
A first irradiation light amount adjusting means which is provided on the optical path of the first irradiation light and adjusts the amount of the first irradiation light;
A first irradiation light polarization means provided on an optical path of the first irradiation light and selecting a polarization direction of the first irradiation light;
Second irradiation light wavelength selection means provided on the optical path of the second irradiation light and selecting the wavelength of the second irradiation light;
A second irradiation light amount adjusting means provided on the optical path of the second irradiation light and adjusting the amount of the second irradiation light;
Second irradiation light polarization means provided on the optical path of the second irradiation light, the transmission polarization axis of which is set perpendicular to the first irradiation light polarization means, and selects the polarization direction of the second irradiation light. When,
The light beam of the first irradiation light whose wavelength is selected, the amount of light is adjusted, and the polarization direction is selected, and the second light beam of the second irradiation light whose wavelength is selected, the amount of light is adjusted and the polarization direction is selected are single. Irradiating light beam synthesizing means for synthesizing the beam,
A mirror that guides the synthesized irradiation light in a direction to irradiate the sample and transmits fluorescence from the sample excited by the irradiation light;
An objective lens disposed between the mirror and the sample;
A fluorescent light beam splitting means for splitting the fluorescent light beam transmitted through the objective lens and the mirror into a first fluorescent light beam and a second fluorescent light beam;
First fluorescence polarization means provided on the optical path of the first fluorescence and having a transmission polarization axis set parallel to the first irradiation light polarization means;
A second fluorescence polarization unit provided on the optical path of the second fluorescence and having a transmission polarization axis set parallel to the second irradiation light polarization unit;
A first imaging means provided on an optical path of the first fluorescence transmitted through the first fluorescence polarization means for capturing a first fluorescence image by the first fluorescence;
A second imaging means provided on the optical path of the second fluorescence transmitted through the second fluorescence polarization means for capturing a second fluorescence image by the second fluorescence;
Image processing means for combining the first fluorescent image and the second fluorescent image;
Image display means for displaying the synthesized fluorescent image;
It has
The first irradiation light intensity adjusting means and the second irradiation light intensity adjusting means can adjust the intensity of the irradiation light by adjusting the intensity of the irradiation light that passes individually. ,
The fluorescence intensity of the first fluorescence and the fluorescence intensity of the second fluorescence are obtained by using at least one of the first illumination light intensity adjustment means, the second illumination light intensity adjustment means, and the image processing means. A microscope image processing apparatus characterized by being equivalent. A white light source,
Irradiation light beam splitting means for splitting the light beam of the irradiation light emitted from the white light source into a light beam of the first irradiation light and a light beam of the second irradiation light;
First irradiation light wavelength selection means provided on the optical path of the first irradiation light and selecting the wavelength of the first irradiation light;
A first irradiation light amount adjusting means which is provided on the optical path of the first irradiation light and adjusts the amount of the first irradiation light;
A first irradiation light polarization means provided on an optical path of the first irradiation light and selecting a polarization direction of the first irradiation light;
Second irradiation light wavelength selection means provided on the optical path of the second irradiation light and selecting the wavelength of the second irradiation light;
A second irradiation light amount adjusting means provided on the optical path of the second irradiation light and adjusting the amount of the second irradiation light;
Second irradiation light polarization means provided on the optical path of the second irradiation light, the transmission polarization axis of which is set perpendicular to the first irradiation light polarization means, and selects the polarization direction of the second irradiation light. When,
The light beam of the first irradiation light whose wavelength is selected, the light amount is adjusted, and the polarization is selected and the light beam of the second irradiation light whose wavelength is selected and the light amount is adjusted and the polarization direction is selected are combined into a single light beam. Irradiating light beam synthesizing means to synthesize,
A first objective lens for guiding the synthesized irradiation light in a direction to irradiate the sample;
A second objective lens on which fluorescence from the sample excited by the irradiation light arranged and synthesized facing the first objective lens across the sample is incident;
A fluorescent light beam splitting means for splitting the fluorescent light beam incident on the second objective lens into a first fluorescent light beam and a second fluorescent light beam;
First fluorescence polarization means provided on the optical path of the first fluorescence and having a transmission polarization axis set parallel to the first irradiation light polarization means;
A second fluorescence polarization unit provided on the optical path of the second fluorescence and having a transmission polarization axis set parallel to the second irradiation light polarization unit;
A first imaging means provided on an optical path of the first fluorescence transmitted through the first fluorescence polarization means for capturing a first fluorescence image by the first fluorescence;
A second imaging means provided on the optical path of the second fluorescence transmitted through the first fluorescence polarization means for capturing a second fluorescence image by the second fluorescence;
Image processing means for combining the first fluorescent image and the second fluorescent image;
Image display means for displaying the synthesized fluorescent image;
It has
The first irradiation light intensity adjusting means and the second irradiation light intensity adjusting means can adjust the intensity of the irradiation light by adjusting the intensity of the irradiation light that passes individually. ,
The fluorescence intensity of the first fluorescence and the fluorescence intensity of the second fluorescence are obtained by using at least one of the first illumination light intensity adjustment means, the second illumination light intensity adjustment means, and the image processing means. A microscope image processing apparatus characterized by being equivalent. The microscope image processing apparatus according to claim 7 or 8, wherein the polarizing means is a polarizing plate or a polarizing beam splitter. The image processing means calculates a luminance ratio between the plurality of fluorescent images;
The microscope image processing apparatus according to claim 1, wherein the image display unit displays an original image and a ratio image of the combined fluorescent image. The microscope image processing apparatus according to any one of claims 1 to 10, wherein the irradiation light amount adjustment unit is insertable into and removable from an optical path of the irradiation light.

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