Microscopic cell observation and inspection system using a plurality of observation methods

The invention relates to a microscopic cell observation and inspection system that uses a total internal reflection cell illuminator that is capable of freely changing an observation position without recourse to any special slide glass, makes sure high SN-ratio observation and facilitates sample manipulation, thereby making high-sensitivity, fast detection of a lot of cell reactions on the same slide glass. While, in response to a command from personal computer (80), step motors (53, 54) are driven to sequentially scan observation positions of cell sample (S) on slide glass (21), one of shutter units (71) and (72) is closed and the other is opened at high speed, whereby either one of illumination optical paths for a TIRF microscope and a drop fluorescence microscope is selected to illuminate cell sample (S) on that observation position. When the drop fluorescence microscope is chosen, filter unit (66) is driven to choose the wavelength of excitation light from drop fluorescence illumination light source (65), so that observation and inspection of cell sample (S) under the TIRF microscope and drop fluorescence microscope can be implemented in an alternate fast switchover way.

ART FIELD

The present invention relates generally to a microscopic cell observation and inspection system using a plurality of observation methods, and more particularly to a microscopic cell observation and inspection system that can combine a total internal reflection fluorescence (TIRF for short) microscope with other microscopes based on optical observation methods such as a confocal fluorescence microscope for fast, high-sensitivity detection of as many as several hundreds of cell reactions on the same slide glass, and can be incorporated into drug design screening apparatus.

BACKGROUND ART

The TIRF microscope technique is a high SN-ratio observation method capable of local excitation on nano-scales. This technique has been widely used for observation of cell membrane activities and single-molecule events in the cell biological field (see Non-Patent Publication 1), and it has made much contributions to experimental revelation of the electrical properties (Non-Patent Publication 1) or Brownian movement of colloidal particles in the electrochemical field as well (Kihm, K. D. et al., Exp. in Fluids, 37, pp. 811-824, (2004). The most noticeable feature of this method is that for fluorescent observation, there are evanescent waves used as an excitation light source, which are generated in association with total internal reflection at an interface of two substances having different refractive indexes.FIG. 1is illustrative of how the evanescent waves are generated; when, on the interface of a substance1having a refractive index n1and a substance2having a refractive index n2, light is incident from the side of the substance2having a higher refractive index at an angle larger than the critical angle, the light is subjected to total internal reflection at that interface. However, there are some evanescent waves unavoidably generated from the interface to the side of the substance1having a lower refractive index, which waves attenuate exponentially. The evanescent waves are light showing up slightly from the total internal reflection interface to an area of about a few tens to a few hundred nm. In the TIRF technique, therefore, evanescent waves are generated at an interface of a sample stained with fluorescent dye and a slide or cover glass (hereinafter called the slide glass) to enable high SN-ratio fluorescent observation at only a limited sample site.

What is now commercially available and generally often used is mainly such an objective lens type TIRF microscope as shown inFIG. 14(a). More specifically, an objective lens of the inverted type is positioned below the slide glass via an oil-immersion oil, and evanescent wave-generation laser light is obliquely incident from below the slide glass via that objective lens so that evanescent waves are generated near the interface with a sample placed on the slide glass. This arrangement performs well and is convenient because the space above the objective lens is freely accessible, and it gives a very bright fluorescent image as well. However, the principal requirement for use of a high-aperture, oil immersion objective lens is supposed to limit observation to one at magnifications of as high as 60 or greater.

A prism type TIRF microscope adapted to enter laser via a prism such as the one shown inFIG. 14(b) has been widely used, too. In this case, a sample is held between two slide glasses with the prism placed on one side glass, and evanescent wave-generation laser light is entered obliquely up in the upper slide glass to generate evanescent waves near the interface of the slide glass in contact with the sample. This arrangement is capable of high SN-ratio observation because of efficient incidence of laser light, and enables low-magnification observation to be easily implemented because of no restriction on magnification at all. However, the space above the objective lens is closed up, resulting in very poor specimen manipulation and more limited degrees of sample flexibility.

As described above, the TIRF microscopes are still far away from meeting the need of making simultaneous comparisons at a plurality of samples of reactions that cells exhibit to various chemicals applied in the process of experimentation. Thus, there is a mounting demand for the development of a TIRF microscope that makes sure good enough specimen manipulation and sample flexibility, facilitates combined use with other optical observation methods, and enables observation at low magnifications.

Past studies include Non-Patent Publication (3) wherein, as shown inFIG. 15(a), an entrance prism and a radiation prism are bonded to the underside of a slide glass; laser light is introduced from the entrance prism into the slide glass where it is subjected to multiple total internal reflection; during that multiple total internal reflection, evanescent waves are generated near the upper surface of the slide glass to excite a specimen; and the laser light guided by multiple total internal reflection goes out via the radiation prism, and Non-Patent Publication 4 wherein, as shown inFIG. 15(b), an end face of a slide glass is processed into an inclined one; laser light is introduced from that inclined end face into the slide glass to subject it to multiple total internal reflection; and upon that multiple total internal reflection, evanescent waves are generated near the upper surface of the slide glass to excite a specimen; and the laser light guided by multiple total internal reflection goes out from the opposite end face of the slide glass. These methods take hold of a free space above the sample and facilitates low-magnification observation, but because the slide glass is limited to a small thickness (0.17 mm in the former, and 0.2 mm in the latter), they offer some problems: a lot more multiple total internal reflections, the occurrence of scattered light due to total internal reflection, attenuation of guided light, the likeliness of the specimen to discolor due to fluorescence, and lower SN ratios. Further, the positions of incidence and radiation of laser light remain fixed, rendering it hard to adjust an optical path for laser light, and making sample manipulation not easy because of the need of moving the objective lens to change a specimen observation position. Yet further, much work is needed with decreased versatility, because of the need of bonding the prisms to the slide glass for each sample.

SUMMARY OF THE INVENTION

In view of such problems with the prior art, the invention has for its object the provision of a microscopic cell observation and inspection system using a plurality of observation methods, which uses a total internal reflection illuminator that enables an observation position to be freely changed on any slide glass without recourse to any special one, makes sure high SN-ratio observation with much more reduced scattered light and facilitates specimen manipulation to make high-sensitivity, fast detection of as many as several hundreds or more of cell reactions on the same slide glass.

According to one aspect of the invention, the aforesaid object is achievable by the provision of a microscopic observation and inspection system using a plurality of observation methods, characterized by comprising a total internal reflection sample illuminator comprising an evanescent wave-generation light source in which laser light from that light source is introduced into a slide glass through which the laser light is guided by multiple total internal reflections to generate evanescent waves on an upper surface of said slide glass so that a cell sample placed on the upper surface of said slide glass is illuminated with said evanescent waves, wherein:

there is a slide glass position adjustment mechanism located, which adjusts a position of said slide glass in a two-dimensional direction along that surface,

a microscope objective lens is located at a position where said slide glass in said total internal reflection sample illuminator is supposed to be placed thereon and in a vertical direction to said slide glass,

on an imaging side of said microscope objective lens, an imaging optical system and an imaging device are located in one optical path via an optical path splitter and an excitation light source for a drop fluorescence microscope is located in another optical path,

shutter units adapted to block off or transmit illumination light are located, one in an optical path from said evanescent wave-generation light source to a laser light inlet of said slide glass, and another between said excitation light source for a drop fluorescence microscope and said optical path splitter,

there is a controller provided which controls an illumination light source switchover by opening or closing each of said shutter units and slide glass position adjustment by said slide glass position adjustment mechanism, and

in response to a command from said controller, said slide glass position adjustment mechanism is controlled to select an observation and inspection position for the cell sample on said slide glass, and in response to a command from said controller, the opening or closing of each of said shutter units is controlled so that an illumination light optical path for said evanescent wave-generation light source and an illumination light optical path for said excitation light source for a drop fluorescence microscope are selectively opened, a total internal reflection fluorescence microscope image and a drop fluorescence microscope image at the selected cell sample observation and inspection site on said slide glass are captured in said controller via said imaging device, and cell reactions are detected from said total internal reflection fluorescence microscope image and said drop fluorescence microscope image.

According to another aspect of the invention, there is provided a microscopic observation and inspection system using a plurality of observation methods, characterized by comprising a total internal reflection sample illuminator comprising an evanescent wave-generation light source in which laser light from that light source is introduced into a slide glass through which the laser light is guided by multiple total internal reflections to generate evanescent waves on an upper surface of said slide glass so that a cell sample placed on the upper surface of said slide glass is illuminated with said evanescent waves, wherein:

said total internal reflection sample illuminator comprises an entrance prism and a radiation prism which support said slide glass movably in a plane thereof via a droplet of index-matching liquid,

said entrance prism is fixed with respect to said light source, and said radiation prism is located in such a way as to be adjustable in terms of position in a direction of travel of said laser light with respect to said entrance prism,

a supporting surface of said entrance prism for said slide glass is flush with a supporting surface of said radiation prism for said slide glass irrespective of a position of the said radiation prism being adjusted,

there is a slide glass position adjustment mechanism provided which takes a grip on said slide glass supported on said entrance prism and said radiation prism to adjust a position of said slide glass in a two-dimensional direction along a plane thereof,

said total internal reflection sample illuminator is set up such that said laser light leaves said supporting surface via said entrance prism and enters said slide glass supported thereon via said index-matching liquid trickling down thereon through which said laser light is guided by multiple total internal reflection, entering from said supporting surface of said radiation prism into said radiation prism via said index-matching liquid trickling down on said supporting surface of said radiation prism, and is radiated out from said radiation prism,

a microscope objective lens is located at a position where said slide glass in said total internal reflection sample illuminator is supposed to be placed between said entrance prism and said radiation prism and in a vertical direction to said slide glass,

on an imaging side of said microscope objective lens, an imaging optical system and an imaging device are located in one optical path via an optical path splitter and an excitation light source for a drop fluorescence microscope is located in another optical path,

shutter units adapted to block off or transmit illumination light are located, one in an optical path from said evanescent wave-generation light source to a laser light inlet of said slide glass, and another between said excitation light source for a drop fluorescence microscope and said optical path splitter,

there is a controller provided which controls an illumination light source switchover by opening or closing each of said shutter units and slide glass position adjustment by said slide glass position adjustment mechanism, and

in response to a command from said controller, said slide glass position adjustment mechanism is controlled to select an observation and inspection position for the cell sample on said slide glass, and in response to a command from said controller, the opening or closing of each of said shutter units is controlled so that an illumination light optical path for said evanescent wave-generation light source and an illumination light optical path for said excitation light source for a drop fluorescence microscope are selectively opened, a total internal reflection fluorescence microscope image and a drop fluorescence microscope image at the selected cell sample observation and inspection site on said slide glass are captured in said controller via said imaging device, and cell reactions are detected from said total internal reflection fluorescence microscope image and said drop fluorescence microscope image.

In a preferable embodiment of the invention, said optical path splitter comprises a dichroic mirror and is operable to prevent light having a wavelength of illumination light from said evanescent wave-generation light source and light having a wavelength of illumination light from said excitation light source for a drop fluorescence microscope from arriving at said optical path on the imaging device side but allow only fluorescence emanating from the cell sample to arrive at said optical path on the imaging device side.

In a preferable embodiment of the invention, between said excitation light source for a drop fluorescence microscope and said optical path splitter there is a filter unit located which selects an excitation wavelength, and in response to a command from said controller an excitation wavelength transmitting through said filter unit is selected.

According to yet another aspect of the invention, there is provided a microscopic observation and inspection system using a plurality of observation methods, characterized by comprising a total internal reflection sample illuminator comprising an evanescent wave-generation light source in which laser light from that light source is introduced into a slide glass through which the laser light is guided by multiple total internal reflections to generate evanescent waves on an upper surface of said slide glass so that a cell sample placed on the upper surface of said slide glass is illuminated with said evanescent waves, wherein:

there is a slide glass position adjustment mechanism located, which adjusts a position of said slide glass in a two-dimensional direction along that surface,

a microscope objective lens is located at a position where said slide glass in said total internal reflection sample illuminator is supposed to be placed thereon and in a vertical direction to said slide glass,

on an imaging side of said microscope objective lens, a filter for blocking off light having a wavelength of illumination light from said evanescent wave-generation light source, a first imaging optical system and a first imaging device are located in one optical path via an optical path splitter, and a confocal scanner for a confocal fluorescence microscope is located in another optical path,

on an illumination side of said confocal scanner an excitation light source for the confocal fluorescence microscope is located, and on an output side of said con-focal scanner a second imaging system and a second imaging device are located,

shutter units adapted to block off or transmit illumination light or fluorescent light are located, one in an optical path from said evanescent wave-generation light source to a laser light inlet of said slide glass, another in an optical path between said confocal scanner and said optical path splitter, and yet another in an optical path between said optical path splitter and said first imaging device,

there is a controller provided which controls an illumination light source switchover by opening or closing each of said shutter units, slide glass position adjustment by said slide glass position adjustment mechanism, and a focus adjustment mechanism adapted to adjust a position of said microscope objective lens in an optical axis direction, and

in response to a command from said controller, said slide glass position adjustment mechanism is controlled to select an observation and inspection position for the cell sample on said slide glass, and in response to a command from said controller, the opening or closing of each of said shutter units is controlled so that an illumination light optical path for said evanescent wave-generation light source and an illumination light optical path for a confocal scanner for said drop fluorescence microscope are selectively opened, and when the illumination optical path for the confocal scanner for said confocal fluorescence microscope is opened, said focus adjustment mechanism is controlled to adjust a position of said microscope objective lens in an optical axis direction to a plurality of given positions, whereby a total internal reflection fluorescence microscope image and a confocal fluorescence microscope image at the selected cell sample observation and inspection position on said slide glass are captured in said controller vial said first imaging device and said second imaging device, respectively, so that cell reactions are detected from said total internal reflection fluorescence microscope image and said confocal fluorescence microscope image.

According to a further aspect of the invention, there is provided a microscopic observation and inspection system using a plurality of observation methods, characterized by comprising a total internal reflection sample illuminator comprising an evanescent wave-generation light source in which laser light from that light source is introduced into a slide glass through which the laser light is guided by multiple total internal reflections to generate evanescent waves on an upper surface of said slide glass so that a cell sample placed on the upper surface of said slide glass is illuminated with said evanescent waves, wherein:

said total internal reflection sample illuminator comprises an entrance prism and a radiation prism which support said slide glass movably in a plane thereof via a droplet of index-matching liquid,

said entrance prism is fixed with respect to said light source, and said radiation prism is located in such a way as to be adjustable in terms of position in a direction of travel of said laser light with respect to said entrance prism,

a supporting surface of said entrance prism for said slide glass is flush with a supporting surface of said radiation prism for said slide glass irrespective of a position of the said radiation prism being adjusted,

there is a slide glass position adjustment mechanism provided which takes a grip on said slide glass supported on said entrance prism and said radiation prism to adjust a position of said slide glass in a two-dimensional direction along a plane thereof,

said total internal reflection sample illuminator is set up such that said laser light leaves said supporting surface via said entrance prism and enters said slide glass supported thereon via said index-matching liquid trickling down thereon through which said laser light is guided by multiple total internal reflections, entering from said supporting surface of said radiation prism into said radiation prism via said index-matching liquid trickling down on said supporting surface of said radiation prism, and is radiated out from said radiation prism,

a microscope objective lens is located at a position where said slide glass in said total internal reflection sample illuminator is supposed to be placed between said entrance prism and said radiation prism and in a vertical direction to said slide glass,

on an imaging side of said microscope objective lens, a filter for blocking off light having a wavelength of illumination light from said evanescent wave-generation light source, a first imaging optical system and a first imaging device are located in one optical path via an optical path splitter, and a confocal scanner for a confocal fluorescence microscope is located in another optical path,

on an illumination side of said confocal scanner an excitation light source for the confocal fluorescence microscope is located, and on an output side of said con-focal scanner a second imaging system and a second imaging device are located,

shutter units adapted to block off or transmit illumination light or fluorescent light are located, one in an optical path from said evanescent wave-generation light source to said entrance prism, another in an optical path between said confocal scanner and said optical path splitter, and yet another in an optical path between said optical path splitter and said first imaging device,

there is a controller provided which controls an illumination light source switchover by opening or closing each of said shutter units, slide glass position adjustment by said slide glass position adjustment mechanism, and a focus adjustment mechanism adapted to a position of said microscope objective lens in an optical axis direction, and

in response to a command from said controller, said slide glass position adjustment mechanism is controlled to select an observation and inspection position for the cell sample on said slide glass, and in response to a command from said controller, the opening or closing of each of said shutter units is controlled so that an illumination light optical path for said evanescent wave-generation light source and an illumination light optical path for a confocal scanner for said drop fluorescence microscope are selectively opened, and when the illumination optical path for the confocal scanner for said confocal fluorescence microscope is opened, said focus adjustment mechanism is controlled to adjust a position of said microscope objective lens in an optical axis direction to a plurality of given positions, whereby a total internal reflection fluorescence microscope image and a confocal fluorescence microscope image at the selected cell sample observation and inspection position on said slide glass are captured in said controller vial said first imaging device and said second imaging device, respectively, so that cell reactions are detected from said total internal reflection fluorescence microscope image and said confocal fluorescence microscope image.

According to the invention, the total internal reflection sample illuminator is set up such that an observation position on any slide glass can be freely changed without recourse to any special one, high SN-ratio observation is enabled with much more reduced scattered light and sample manipulation is facilitated; it is possible to make high-sensitivity, fast observation and inspection of as many as several hundreds or more of cell reactions on the same slide glass using a plurality of observation methods such as a drop fluorescence microscope technique and a confocal fluorescence microscope technique, and make a lot of contributions to drug design screening, and so on.

BEST MODE FOR CARRYING OUT THE INVENTION

The microscopic cell observation and inspection system using a plurality of observation methods according to the invention is now explained with some embodiment and examples. Before that, the total internal reflection sample illuminator—the most important part of the microscopic cell observation and inspection system of the invention—is explained with reference to its example.FIG. 1is a vertical sectional view of one example of the total internal reflection sample illuminator used with the microscopic cell observation and inspection system according to the invention andFIG. 2is a plan view of that. This total internal reflection sample illuminator comprises a sample table1with an opening2provided in its center. Facing the opening2, an entrance prism3is fixed to the sample table1. Facing the entrance prism3, a radiation prism4is attached to a moving table5with the opening2held between them such that the space between the radiation prism4and the entrance prism3is adjustable. This moving table5is movable and adjustable by a position adjustment screw6toward the entrance prism3with respect to the sample table1. And the entrance prism3and the radiation prism4are configured and the moving table5has a moving mechanism set up such that irrespective of where the moving table5is positioned, the transmissive surface33of the entrance prism3is flush with the transmissive surface41of the radiation prism4.

And a slide glass for supporting a cell sample S, for which a slide glass chamber20is here used, is freely placed over the transmissive surface33of the entrance prism3and the transmissive surface41of the radiation prism4via an immersion oil25trickling down to the transmissive surfaces33and41.

The entrance prism3here has a reflection prism configuration comprising a transmissive surface31through which evanescent wave-generation laser light10is entered from a laser11via a reflecting mirror12, a reflective surface32adapted to reflect the incident laser light10, and a transmissive surface33through which the laser light10reflected off at the reflective surface32goes out, and the radiation prism4has a transmission prism configuration comprising a transmissive surface41adapted to guide to the outside of the slide glass21laser light10guided by multiple total internal reflections through the slide glass21at the bottom of the slide glass chamber20, and a transmissive surface42adapted to allow the laser light10incident from the transmissive surface41to radiate out.

The sample table1is also provided on its upper surface with a slide glass grip mechanism55via an X-Y stage50. In the example here, the slide glass grip mechanism55supports the slide glass chamber20by means of elasticity acting in the direction that it is sandwiched between pads56; by an X-direction position adjustment screw51at the X-Y stage50, the slide glass chamber20is movable and adjustable in the X-direction parallel with the incidence direction of the laser light10; and by a Y-direction position adjustment screw52at the X-Y stage50, the slide glass chamber20is movable and adjustable in the Y-direction at right angles with the incidence direction of the laser light10. And the rotation of the X-direction position adjustment screw51, and the Y-direction position adjustment screw52is controlled by a step motor53, and54to only a given amount, respectively.

The arrangement being like such, even when the slide glass chamber20or slide glass21placed over the transmissive surfaces33and41of the entrance prism3and radiation prism4via the immersion oil25is replaced by a different or standard type, there is no need of adjusting the entrance position of the evanescent wave-generation laser light10whatsoever, and there is no need of adjusting the position of the laser11, either, that provides a light source for it. Note, however, that a different thickness of the slide glass21causes a change in the reflection position at the bottom surface of the slide glass21. If, in this case, the position adjustment screw6is adjusted to move the moving table5in the X-direction, it is then possible to optimize the position of the radiation prism4thereby preventing scattered light from occurring.

And the step motors53and54are rotated a given amount to adjust the X- and Y-direction position adjustment screws51and52at the X-Y stage50to move the slide glass grip mechanism55arbitrarily within the plane defined by the transmissive surfaces33and41of the entrance prism3and radiation prism4so that the position of the slide glass chamber20in that plane is arbitrarily moved and adjusted, whereby it is possible to move and adjust the position of the cell sample S under observation on the slide glass21in the orthogonal direction to the optical axis of a microscope objective lens61facing the center opening in the sample table1.

The total internal reflection sample illuminator used with the microscopic cell observation and inspection system of the invention—explained with reference to the aforesaid example—is common to the prior art multiple total internal reflection type shown inFIGS. 15(a) and15(b) in that the laser light is subjected to multiple total internal reflections within the slide glass21. If a slide glass thicker than an ordinary thickness of 0.17 to 0.2 mm is used as the slide glass21, however, it is then possible to minimize the number of times of multiple total internal reflections, thereby preventing a drop of SN ratios upon fluorescent observation. In the aforesaid example, the number of times of multiple total internal reflections on the upper surface of the slide glass21with the sample S placed on it is four: there are four discrete areas illuminated with evanescent waves Ev, as shown inFIG. 2.

It is also possible to optimize the position of the radiation prism4thereby preventing scattered light from resulting from the laser light10that leaves the slide glass21after multiple total internal reflections.

And the sample is easily manipulated from above, and combined use with other optical observation methods and low-magnification observation at high SN ratios are viable as well. The sample container used may be a slide glass chamber for cell culture such as the aforesaid one, and there is no need of processing slide glasses at all, leading to improved versatility.

Further, the observation position on the slide glass21is changeable arbitrarily, freely and fast via the position adjustment mechanism at the X-Y stage50, so that a lot of cell samples S positioned within the plane of the slide glass21can be observed in a fast switchover way.

It is here noted that when there is a change in the thickness of the slide glass21, there is also a change in the reflection position on the upper surface of the slide glass21. Consequently, the area illuminated with the evanescent waves Ev is off the optical axis of the microscope objective lens61. Therefore, to implement observation when there is a change in the thickness of the slide glass21, any of the sample table1, objective lens61and reflecting mirror12is moved and adjusted in the X-direction in association with a shift of the position irradiated with the evanescent waves Ev.

Now then, the invention uses the total internal reflection sample illuminator of such structure as mentioned above to implement observation of the same cell sample S on the slide glass21under a TIRF microscope, a drop fluorescence microscope or a confocal fluorescence microscope in a fast switchover way.

FIG. 3is illustrative of one example of that, i.e., the construction of the microscopic cell observation and inspection system that can be used in combination with observations under the TIRF microscope and drop fluorescence microscope. That construction is now explained.

There is an opening2formed in the center of the sample table1ofFIGS. 1 and 2, under which there is a microscope objective lens61of the inverted type located, and there is a dichroic mirror62located obliquely located in an optical path through that microscope objective lens61on the observation side. And there is an imaging lens63located in an optical path of light transmitting through the dichroic mirror62, and an imaging device64such as a CCD is located in the imaging plane.

On the other hand, a drop fluorescent illumination light source65such as a xenon lamp is located on the incidence side of the obliquely disposed dichroic mirror62. And between the drop fluorescence illumination light source65and the dichroic mirror62there is an excitation wavelength select filter unit66located.

Further, a shutter unit71for blocking off or transmitting laser light10is disposed in an optical path between the entrance prism3and the evanescent wave-generation laser light11in the total internal reflection sample illuminator ofFIGS. 1 and 2, and between the drop fluorescence illumination light source65and the dichroic mirror62there is a shutter unit72for blocking off or transmitting drop fluorescence excitation light.

Between the dichroic mirror62and the imaging lens63, there is a barrier filter62for blocking off fluorescence of unwanted wavelengths, etc.

And the aforesaid shutter units71,72, filter unit66, and step motors53,54(FIG. 2) for moving and adjusting the observation position of the cell sample S on the slide glass21are connected to a personal computer80, and switchover or positions of them are controlled by a command from the personal computer80. Fluorescent images of the cell sample S taken by the imaging device64based on such operations are captured in the personal computer80for given image processing.

The microscopic cell observation and inspection system ofFIG. 3being arranged like such, while the step motors53,54are driven in response to a command from the personal computer80to sequentially scan the observation positions of the cell sample S on the slide glass21, one of the shutter units71and72is closed and another opened at high speed, whereby either one of the optical paths through the TIRF microscope and drop fluorescence microscope is selected to illuminate the cell sample S on that observation position. When the drop fluorescence microscope is chosen, the filter unit66is driven to choose the wavelength of excitation light from the drop fluorescent illumination light source65, whereby the observation and inspection of the cell sample S under the TIRF microscope and drop fluorescence microscope can be implemented in a fast switchover way.

In addition, the total internal reflection sample illuminator used with the microscopic cell observation and inspection system of the invention can make use of a generally available slide glass chamber or slide glass without recourse to any special ones, and change the cell sample S to be observed and inspected on the slide glass chamber20or slide glass21to an arbitrary position at a fast speed without any movement of the light source laser11or microscope objective lens61; for instance, if a chemical is added to the cell sample S on the slide glass21in a varying concentration or kind depending on position, it is then possible to make sure rapid detection of the efficacy of the chemical, etc. on the same cultured cell.

Examples of detection using the microscopic cell observation and inspection system ofFIG. 3are now explained.

FIG. 4is illustrative in schematic of results of observation under drop fluorescent illumination of a cell with the genetic introduction in it of a fused protein of protein kinase Cα (PKCα) that is one of polyfunctional enzymes and green fluorescent protein (GFP). As shown inFIGS. 4(a) and4(b), PKCα localizes from within cytoplasm C to a cell membrane PM upon receipt of depolarization stimulation, at which it is activated. Arrows directing from the nucleus N ofFIG. 4(b) toward radiation directions are the directions of localization of PKCα, and hatched portions ofFIG. 4are indicative of sites where the concentration of PKCα is high. InFIGS. 4(a) and4(b), the upper is a plan view and the lower a side view. With a change of this localization as an index to PKC activation, PKC activation could be monitored over time as a change in the spatial fluorescence intensity of GFP in a living cell.

The aforesaid phenomenon is two-dimensionally observed under a drop fluorescence microscope in the form of migration of green fluorescence from the upper drawing ofFIG. 4(a) to the upper drawing ofFIG. 4(b). Fluorescence intensity changes are viewed in the form of changes in the opposite directions: a decrease in the intensity of green fluorescence at the cytoplasm C and an increase in the intensity of green fluorescence at the cell membrane PM.

Under a TIRF microscope, by contrast, the same phenomenon is viewed in the form of migration of green fluorescence from the lower drawing ofFIG. 4(a) to the lower drawing ofFIG. 4(b): in the form of an exponential increase in the intensity of fluorescence of GFP near the lower cell membrane PF of the cell (the range of about 100 nm from the glass surface at which the evanescent field occurs), which leads to much more improved SN ratios.

FIGS. 5 and 6are indicative of changes in images viewed under the drop fluorescence microscope and TIRF microscope, respectively, corresponding to the upper and lower drawings ofFIG. 4. More specifically,FIGS. 5(a),5(b) and5(c) andFIGS. 6(a),6(b) and6(c) are views illustrative of experimental examples with the introduction in living cells of ions K+for depolarization stimulation. A graph at the right side ofFIG. 5, andFIG. 6is indicative of changes in the intensity of fluorescence in the area of interest (the rectangular area in (a)), referring to the same phenomenon. However, it is found that there is an about ten-fold difference in the amount of change depending on the difference in the measuring methods (detections under the drop fluorescence microscope and TIRF microscope).

By the way, the concentration of calcium in a calcium sensitive dye Fura 2 is measured in the form of the ratio of change in the intensity value of fluorescence near 510 nm that is a fluorescence wavelength as Fura 2 is excited at two wavelengths of 340 nm and 380 nm.FIG. 7is illustrative of the calcium concentration of Fura 2 vs. fluorescence characteristics with respect to excitation wavelengths. InFIG. 7, Em is a fluorescence wavelength. That is, as there is a rise in the concentration of calcium in a cell due to depolarization stimulation, there is a decrease in the 510 nm fluorescence wavelength of Fura 2 loaded into the cell excited at 380 nm, whereas there is rather an increase in the 510 nm fluorescence wavelength of Fura 2 excited at 340 nm. Therefore, as there is a rise in the concentration of calcium in the cell, there is an increase in the value of (510 nm fluorescence intensity of Fura 2 excited at 340 nm/510 nm fluorescence intensity of Fura 2 excited at 380 nm).

FIG. 8is illustrative of changes over time of values found by alternate measurement at a one-second interval of changes in the fluorescence intensity of Fura 2 at two ultraviolet excitation wavelengths using the drop fluorescence microscope ofFIG. 3, and changes in the fluorescence intensity of GFP using the TIRF microscope ofFIG. 3. InFIG. 8, TEA is a chemical (tetraethyl-ammonium) added for the purpose of depolarization stimulation.

That is, inFIG. 3, the step motors53,54are first driven in response to a command from the personal computer80to select a specific observation position for the cell sample S on the slide glass21. Then, the shutter unit71is opened with the shutter unit72remaining closed to select the illumination optical path for the TIRF microscope. Then, the cell sample S at that observation position is illuminated with the evanescent wave Ev generated while the laser light10is subjected to multiple total internal reflections through the slide glass21. Finally, a PKCα-GFP fluorescent image that is a fluorescent image of that sample is formed on the imaging device64by the imaging lens63via the microscope objective lens61and dichroic mirror62, following by capturing the ensuing image in the personal computer80.

Then, the shutter unit71is closed and the shutter unit72is opened and, at the same time, the filter unit66is driven to select 340 nm out of the wavelengths of excitation light75from the drop fluorescence illumination light source65. Then, the cell sample S at that observation position is illuminated with that excitation light75via the microscope objective lens61in a drop illumination way. Finally, fluorescence76from that observation position is imaged on the imaging device64by the imaging lens63via the microscope objective lens61and dichroic mirror62, followed by capturing a fluorescent image of 510 nm fluorescence wavelength excited with 340 nm excitation light in the personal computer80.

Then, while this time the shutter unit71remains closed and the shutter unit72remains opened, the filter unit66is driven to select 380 nm of the excitation light75from the drop fluorescence illumination light source65so that the cell sample S at that observation position is illuminated with the excitation light75via the microscope objective lens61in a drop illumination way. Finally, fluorescence76from that observation position is imaged on the imaging device64by the imaging lens63via the microscope objective lens61and dichroic mirror62, followed by capturing a fluorescent image of 510 nm fluorescence wavelength excited with 340 nm excitation light in the personal computer80.

This process is repeated for a specific observation position whereby there is such a change over time as shown inFIG. 8obtained.

By measuring the behaviors of quite different parameters (polyfunctional enzyme and calcium concentration), it is thus possible to enhance the sensitivity and specificity of the cell to reactions. Referring again toFIG. 8, it is noted that as the calcium concentration rises, there is a change in the localization of PKCα. However, it is not that the increase in the calcium concentration necessarily leads to the change in the localization of PKCα: the aforesaid phenomenon is going to occur depending on cell states, the concentration and type of the chemical added, etc.

FIGS. 9(a),9(b) and9(c), corresponding toFIG. 8, are illustrative of changes in images under the TIRF microscope (a), the drop fluorescence microscope at 340 nm excitation wavelength (b), and the drop fluorescence microscope at 380 nm excitation wavelength, with the left being images before depolarization stimulation (resting) and the right being images upon depolarization stimulation (stimulation). Changes in the rectangles are found to be in coincidence with those in the foregoing explanation.

Referring then toFIG. 10, it is illustrative of the construction of another example of the microscopic cell observation and inspection system that may be used in combination with observations under a TIRF microscope and a confocal fluorescence microscope. The arrangement here is now explained.

As is the case with the example ofFIG. 3, the opening2is provided in the center of the sample table1, and below that there is a microscope objective lens61of the inverted type located. A half-mirror81is obliquely located in an optical path on the objection side of that microscope objective lens61. And an excitation light cut filter82, a shutter unit85and an imaging lens63are located in a path taken by light transmitting through the half-mirror81, and an imaging device64such as a CCD is located on the imaging plane of that imaging lens63.

On the reflection side of the obliquely located half-mirror8, on the other hand, there are a Nipkow type confocal scanner90and a laser120for illuminating it located via a relay lens84, and on the output side of the confocal scanner90there is an imaging lens129located, with an imaging device151such as a CCD located on the imaging plane of that imaging lens.

The microscope objective lens61here is adjustable by a piezo element86in terms of position in the optical axis direction.

Further, in an optical path between the entrance prism3and the laser11for generating the evanescent wave-generation laser light10in the total internal reflection sample illuminator ofFIGS. 1 and 2, there is a shutter unit71provided for blocking off or transmitting the laser light10, and between the confocal scanner90and the half-mirror81, there is a shutter unit83located for blocking off or transmitting confocal scan light.

Here, when the shutter unit83stays open, the laser light or excitation light generated from the laser120passes through the Nipkow type confocal scanner90and relay lens84, turning into parallel scan light, which is in turn reflected off at the half-mirror81. The reflected light then converges onto a given object surface in the cell sample S on the slide glass21via the microscope objective lens61. Fluorescence emitted out of the cell sample S excited by the laser light comes back to the confocal scanner90via the microscope objective lens61, half-mirror81and relay lens84, and is reflected off at a dichroic mirror124in the cofoncal scanner90, imaging an fluorescent image of the given object surface in the cell sample S on an imaging device151through an imaging lens129.

The Nipkow type confocal scanner90, for instance, has such construction as shown inFIG. 11. InFIG. 11, laser light121or excitation light from a laser120is converged into individual light beams by means of individual microlens123located on a microlens disk122, and after transmitting through the dichroic mirror124, those light beams pass through individual pinholes126formed in a pinhole disk (also called the Nipkow disk)125, coming together at the given object surface in the cell sample S via the relay lens84and microscope objective lens61.

Fluorescence given out of the cell sample S again passes through the microscope objective lens61, half-mirror81(FIG. 10) and relay lens84, coming together on the individual pinholes126in the pinhole disk125. Fluorescence transmitting through the individual pinholes126is reflected off at the dichroic mirror124, imaging a fluorescent image on the imaging device151such as CCD in the imaging unit by means of the imaging lens129.

The dichroic mirror124used here is designed in such a way as to transmit excitation light121and reflect fluorescence from the cell sample S.

The microlens disk122is mechanically coupled by a member127to the pinhole disk125so that they rotate around a rotary shaft128. The individual microlenses123and pinholes126are located such that the plane of the cell sample S under observation is scanned by excitation light from the individual pinholes126formed in the pinhole disk125. The plane with the pinholes126lined up on it, the plane of the cell sample S under observation and the imaging device151in the imaging unit are located in mutually optically conjugate relations so that optical section images, viz., confocal images of the cell sample S are formed on the imaging device151.

And the foregoing shutter units71,83,85, Nipkow type confocal scanner90, piezo element86for adjusting the position of the object plane (plane under observation) of the microscope objective lens61relative to the cell sample S, and step motors53,54(FIG. 2) for moving and adjusting the observation position of the cell sample S on the slide glass21are connected to the personal computer80, and their switchover or position control is implemented in response to a command from the personal computer80. Fluorescent images of the cell sample S taken by the imaging device64,151based on their operations are captured in the personal computer80for the purpose of implementing the necessary imaging processing.

The microscopic cell observation and inspection system ofFIG. 10being constructed like such, the step motors53,54are driven in response to a command from the personal computer80to sequentially scan the observation positions of the cell sample S on the slide glass21. In the meantime, one of the shutter units71and82and the shutter unit83is fast closed and another is opened, whereby one of the observation optical paths through the TIRF and confocal fluorescence microscopes is selected to illuminate the cell sample S at the position under observation. When the confocal fluorescence microscope is chosen, the piezo element86is controlled to adjust the position of the microscope objective lens61in the optical axis direction at a video rate so that confocal planes are varied by a predetermined number to take a florescent image of each cell section by the imaging device151. Finally, the fluorescent image of each section is captured in the personal computer80to construct a three-dimensional fluorescent image of the cell sample S.

When there is the observation optical path for the TIRF microscope chosen, the cell sample S at that observation position is illuminated with evanescent waves Ev generated while the laser light10is subjected to multiple total internal reflections in the slide glass21, and the ensuing fluorescent image is imaged by the imaging lens63and taken by the imaging device64via the microscope objective lens61, half-mirror81and excitation light cut filter82, and then captured in the personal computer80.

In this way, for instance, each section in the cell is imaged by the confocal fluorescence microscope and sites near the cell membrane are imaged by the TIRF microscope so that the behaviors of a protein mass migrating in cell organelles and cytoplasm and between cell membranes can be viewed using fluorescent protein.

In the examples described above, the excitation light source or the lasers11,120are supposed to have a single wavelength, and the fluorescence wavelength is supposed to be a single one as well. However, it is understood that a laser combiner may be used to superpose a plurality of excitation wavelengths one upon another over time or at the same time or, alternatively, to take fluorescent images having different wavelengths corresponding to the respective excitation wavelengths, a fluorescence separation unit may be disposed in front of a CCD camera (imaging lens63plus imaging device64, or imaging lens129plus imaging device151) or a color CCD camera may be used to take a multicolor image.

An example of detection using the microscopic cell observation and inspection system ofFIG. 10is now explained: a plurality of fluorescence wavelength images can be simultaneously taken.

FIG. 12is illustrative in schematic of the behavior of a glucose transporter. The glucose transporter works as a sort of a device of taking sugar in an in-vivo cell. In a muscle or fat, glucose transporter4(Glut 4) is incorporated in its cell and, as shown inFIG. 12, and it is delivered by granules from the Golgi body of the cell organelle. As a lot of Glut 4's are localized from within the cell to the cell membrane via insulin stimulation, it permits the rate of taking sugar in to increase. Here, as shown inFIG. 12, a fused protein of Glut 4 and green fluorescent protein (GFP) is genetically introduced, a recognition antibody capable of recognizing that Glut 4 is previously labeled with a red dye (alexa 568 or the like), and a glucose transporter recognition antibody is disposed outside the cell. As Glut 4 shows up on the cell membrane by insulin stimulation, the fluorescence intensity of GFP rises, and the recognition antibody labeled with the red dye is bonded to Glut 4: that Glut 4 has indeed been migrated onto the cell membrane is made certain under the TIRF microscope. With that, how Glue4returns again within the cell is also observable under the confocal fluorescence microscope. Where Glut 4 goes out is comparable with where Glut 4 goes back to. In that case, at the TIRF plane with evanescent waves existing on it, the bond of Glut 4 with the recognition antibody can be confirmed and observed. Further, the behavior of Glut 4migrating onto the cell membrane can be observed under the confocal fluorescence microscope, and the behavior of Glut 4 going back from the cell membrane is observed on, for instance, confocal planes1to10or the like. The apparatus ofFIG. 10is useful for the continuous taking of such fluorescent images.

It is noted that the apparatus ofFIG. 3may be combined with the apparatus ofFIG. 10(for instance, by locating the half-mirror81between the microscope objective lens61and the dichroic mirror62inFIG. 3) to enable combined use of observations under the TIRF microscope, drop fluorescence microscope and confocal fluorescence microscope.

With the aforesaid microscopic cell observation and inspection system of the invention using a plurality of observation ways, it is possible to observe and inspect nano-scale phenomena in association with a plurality of cells or bio-high molecules at low magnifications and at the same time. The system enables changes in the localization of organelles, protein delivery and protein in a lot of cells to be observed and inspected at the same time or almost simultaneously, so that morphological comparison, analysis of protein delivery mechanism, and detection of enzyme activity can be facilitated, making a great contribution to studies in the cell biological field and drug design screening. Further, since commercial slide glass chambers for cell culture are used for sample containers, the inventive system is of by far greater versatility. Still further, since as many as several hundreds of cell reactions are detectable on the same slide glass with high sensitivity at high speed, the system of the invention may be used as a drug design screening system.