Culture microscope apparatus

A culture microscope apparatus has an illumination unit to apply excitation light to the specimen, a specimen observing portion to observe light generated from the specimen due to the excitation light, and a dimmer unit to dim the excitation light that has penetrated the specimen.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based upon and claims the benefit of priority from prior Japanese Patent Applications No. 2003-365025, filed Oct. 24, 2003; and No. 2004-142635, filed May 12, 2004, the entire contents of both of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a culture microscope apparatus to observe living cells that have been kept alive under a constant environmental condition for long-term observation of cells of living organisms such as animals or plants.

2. Description of the Related Art

Generally, in such fields as biochemistry, living cells of animals or plants are kept alive under a suitable condition to observe behavior of the living cells for functional analysis of living organisms.

For the behavioral observation of living cells, fluorescent dyes using antigen antibody reactions and fluorescent observation using gene-transferred fluorescent protein are utilized. The fluorescent observation is used because it enables observation on the molecular level and behavioral observation of distributions and molecules. It is to be noted here that the fluorescence is a phenomenon in which if energy such as ultraviolet rays is applied to a substance from the outside, atoms of the substance transit from ground state to excited state and then emit specific light when returning to the ground state. The ultraviolet rays from the outside are generally called excitation light.

On the other hand, there is a phenomenon called discoloration in which if the fluorescent dyes and the fluorescent protein are continuously exposed to the excitation light, the intensity of fluorescence light is gradually lowered or nullified. Therefore, the fluorescent observation requires attention, for example, using as weak excitation light as possible and exposing a sample to the excitation light during observation only.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed, according to an aspect of the invention, to a culture microscope apparatus that reduces discoloration of fluorescent dyes. The culture microscope apparatus according to the present invention comprises illumination means for applying excitation light to a specimen, specimen observing means for observing light generated from the specimen due to the excitation light, and dimmer means for dimming the excitation light that has penetrated the specimen.

The present invention is directed, according to another aspect of the invention, to a culture microscope apparatus that has less temperature changes in an installation environment and is easily cleaned. The culture microscope apparatus according to the present invention comprises a microscope device to observe a specimen, a culture device capable of controlling temperature and humidity, and isolation means for isolating the microscope device from moisture of the culture device.

DETAILED DESCRIPTION OF THE INVENTION

First Embodiment

FIG. 7shows a schematic configuration of a conventional culture microscope apparatus disclosed in Jpn. Pat. Appln. KOKAI Publication No. 2003-93041. In this culture microscope apparatus, living samples contained in culture cases1503placed on sample tables1502in a culture container1501are cultured in the culture container1501, and images of the living samples are picked up by a CCD1504for observation. In order to observe fluorescent images of the living samples, light from a fluorescence measurement excitation light source1505is projected from an excitation light projection fiber1506, reflected by mirrors1507and1508, and applied as excitation light through objective lens1509to the living sample in the culture case1503, and then an image of fluorescence light emitted from the living sample is picked up by the CCD1504through the objective lens1509. On the other hand, for observation of transmitted light image of the living sample, light from a white light source1510is applied to the living sample in the culture case1503through a white light projection fiber1511, and an image of light that has penetrated the living sample is picked up by the CCD1504through the objective lens1509.

In the culture microscope apparatus of Jpn. Pat. Appln. KOKAI Publication No. 2003-93041, most of the excitation light that has been projected from the excitation light projection fiber1506and applied through the objective lens1509to the living sample in the culture case1503may penetrate the culture case1503and be reflected by the surface of a fixed arm1512supporting an projection end of the fiber1511, so that the reflected excitation light might excite the living samples that are not targeted for observation to fade fluorescent dyes. Further, even if all the excitation light that has penetrated the culture case1503can be captured by the white light projection fiber1511, light reflected by a lamp of the white light source1510or the like may return through the white light projection fiber1511to fade the fluorescent dyes.

In view of such circumstances, the present embodiment is directed to a culture microscope apparatus that reduces the discoloration of the fluorescent dyes.

FIG. 1shows a schematic configuration of the culture microscope apparatus to which the present invention is applied, andFIG. 2shows an enlarged schematic configuration of essential parts of the culture microscope apparatus.

A culture microscope apparatus body1101is provided with a culture container1102. The culture container1102is provided with multistage (three stages in an example shown) culture case support racks1104. Each of the culture case support racks1104supports (three in an example shown) specimens (i.e., culture cases1103containing living cells1129). The culture case support rack1104is provided with a recess1104aslightly larger than the culture case1103, and an opening1104bslightly smaller than the culture case1103on a bottom surface of the recess1104a, as shown inFIG. 2. Thus, the culture case support rack1104uses the recess1104ato position the culture case1103and allows of observation of the living cells1129in the culture case1103through the opening1104b.

Here, the culture case1103comprises, for example, a dish and a micro well-plate to which a culture solution is added, and a necessary amount of living cells1129is distributed and contained in the culture case1103. Further, the culture case1103is made of a resin material to which a colorless and transparent glass is affixed or in which a glass is affixed on its bottom surface.

A valve1135is connected to the culture container1102. This valve1135is for supplying carbon dioxide into the culture container1102. A temperature adjustment heater1137for temperature adjustment and an evaporating dish1136retaining water are located on the bottom surface of the culture container1102, and a humidification heater1140is located under the evaporating dish1136. Further, a sensor1141is provided in the culture container1102. This sensor1141is intended to detect carbon dioxide concentration, humidity and temperature in the culture container1102.

A controller1138is connected to the valve1135, the temperature adjustment heater1137, the humidification heater1140and the sensor1141. The controller1138is intended to perform various kinds of control; for example, if the carbon dioxide concentration, humidity and temperature in the culture container1102are detected by the sensor1141, the controller1138opens the valve1135in accordance with an output of the detection to supply carbon dioxide, turns on the humidification heater1140to evaporate water from the evaporating dish1136for supply of steam, or turns on the temperature adjustment heater1137to supply heat for adjustment of temperature in the culture container1102. This maintains a constant environmental condition in the culture container1102necessary for the living cells1129so that the living cells1129can stay alive for a long period of time in the culture container1102.

On the other hand, a pair of first guides1105and a first movement screw1106are arranged on the bottom surface of the culture container1102in parallel in a direction perpendicular to the drawing.

The first guide1105has a fixed portion1105aand a moving portion1105b, and the moving portion1105bis linearly movable in contrast with the fixed portion1105a. The first movement screw1106has a screw portion1106aand a nut portion1106b, and the screw portion1106ais turned while the rotation of the nut portion1106bis regulated so that the nut portion1106bmoves straight.

A first moving part1107is attached to the moving portion1105bof the first guide1105and the nut portion1106bof the first movement screw1106. Further, a first drive portion1109is attached to the screw portion1106aof the first movement screw1106. The first drive portion1109rotationally drives the screw portion1106aof the first movement screw1106under instructions from the controller1138, and the rotation of the screw portion1106ais converted to straight movement of the nut portion1106b, thereby enabling the first moving part1107to move in a direction (direction perpendicular to the drawing) guided by the first guide1105.

In the first moving part1107, a pair of guide mounting surfaces1107a,1107bis provided to face each other perpendicularly to the bottom surface of the culture container1102. The guide mounting surface1107ais provided with a second guide1110, and the guide mounting surface1107bis provided with a second movement screw1108. The second guide1110and the second movement screw1108are configured in the same manner as the first guide1105and the first movement screw1106. In other words, the second guide1110has a fixed portion1110afixed to the guide mounting surface1107aand a moving portion1110b, and allows the moving portion1110bto move linearly relative to the fixed portion1110a. Further, the second movement screw1108has a screw portion1108afixed to the guide mounting surface1107aand a nut portion1108b, and the screw portion1108ais turned so that the nut portion1108bmoves straight.

A second moving part1111is attached to the moving portion1110bof the second guide1110and the nut portion1108bof the second movement screw1108. Further, a second drive portion1139is attached to the screw portion1108aof the second movement screw1108. The second drive portion1139rotationally drives the screw portion1108aof the second movement screw1108under instructions from the controller1138, and the rotation of the screw portion1108ais converted to straight movement of the nut portion1108b, thereby enabling the second moving part1111to move in a direction (vertical direction of the drawing) guided by the second guide1110.

The second moving part1111is made from a rectangular frame, and has longitudinal portions1111a,1111balong the guide mounting surfaces1107a,1107band guide support portions1111c,1111dparallel with the culture case support rack1104.

The guide support portion1111cis provided with a third guide1113and a third movement screw1114, and the guide support portion1111dis provided with a fourth guide1115and a fourth movement screw1116. The third guide1113and the fourth guide1115are configured in the same manner as the first guide1105. The third movement screw1114and the fourth movement screw1116are also configured in the same manner as the first movement screw1106. Here, the third movement screw1114has a screw portion1114afixed to the guide support portion1111cand a nut portion1114b, and the screw portion1114ais turned so that the nut portion1114bmoves straight. The fourth movement screw1116also has a screw portion1116ato the guide support portion1111dand a nut portion1116b, and the screw portion1116ais turned so that the nut portion1116bmoves straight.

Specimen observing means or a specimen observing portion1121is attached to the nut portion1114bof the third movement screw1114. Further, a third drive portion1117is attached to the screw portion1114aof the third movement screw1114. The third drive portion1117rotationally drives the screw portion1114aof the third movement screw1114under instructions from the controller1138, and the rotation of the screw portion1114ais converted to straight movement of the nut portion1114b, thereby enabling the specimen observing portion1121to move in accordance with the third guide1113in a direction (horizontal direction of the drawing) along the culture case support rack1104.

Dimmer means or a dimmer unit1122is attached to the nut portion1116bof the fourth movement screw1116. Further, a fourth drive portion1118is attached to the screw portion1116aof the fourth movement screw1116. The fourth drive portion1118rotationally drives the screw portion1116aof the fourth movement screw1116under instructions from the controller1138, and the rotation of the screw portion1116ais converted to straight movement of the nut portion1116b, thereby enabling the dimmer means or dimmer unit1122to move in accordance with the fourth guide1115in a direction (horizontal direction of the drawing) along the culture case support rack1104.

In this case, the controller1138simultaneously drives the third movement screw1114by the third drive portion1117and the fourth movement screw1116by the fourth drive portion1118, and controls so that the specimen observing portion1121faces the dimmer means or dimmer unit1122with the culture case1103put between them and an optical axis X of the specimen observing portion1121coincides with an optical axis Y of the dimmer means or dimmer unit1122.

The specimen observing portion1121is connected with Illumination means or an illumination unit. The illumination means or illumination unit includes an incident-light fiber1124and an external illumination portion1123. The specimen observing portion1121is connected to the external illumination portion1123through the incident-light fiber1124. The external illumination portion1123comprises a mercury lamp, a xenon lamp, a laser light source or the like.

The specimen observing portion1121has, as an observation optical system to observe the living cells1129in the culture case1103, a first lens1134, an excitation filter1125, an objective lens1131, a dichroic mirror1127, an emission filter1126and an image-forming lens1144, and also has image pickup means or an image pickup device1128such as a CCD. Here, the excitation filter1125has properties that only transmit wavelengths necessary for the excitation of the fluorescent dyes. The dichroic mirror1127has properties that reflect the excitation light having a relatively short wavelength and transmit the relatively fluorescence light having a long wavelength. Moreover, the emission filter1126has properties that selectively transmit the fluorescence light emitted by the fluorescent dyes.

Furthermore, if illumination light from the external illumination portion1123is introduced to the specimen observing portion1121through the incident-light fiber1124, the light projected from the incident-light fiber1124will be light converging on a rear focal plane of the objective lens1131through the first lens1134, and the light is selected by the excitation filter1125, reflected by the dichroic mirror1127and converges on the rear focal plane of the objective lens1131. Further, the light converging on the rear focal plane of the objective lens1131is brought into parallel light by the objective lens1131to illuminate an illumination range uniformly as excitation light1142that excites the fluorescent dyes in the living cells1129in the culture case1103. The fluorescent dyes excited by the excitation light1142emit fluorescence light1143having a wavelength longer than that of the excitation light1142. The fluorescence light1143is brought into parallel light by the objective lens1131, penetrates the dichroic mirror1127and the emission filter1126, and falls on an image pickup plane of the image pickup device1128through the image-forming lens1144, thereby picking up an image thereof.

Meanwhile, the illumination range is dotted with the living cells1129, and parts to be observed within the living cells1129are further limited and less. Thus, most of the excitation light1142in the illumination range penetrates the culture case1103to arrive at the dimmer means or dimmer unit1122.

The dimmer means or dimmer unit1122has a sealing glass1132, a reflective member1122aand a light absorption member1122bto dim the excitation light penetrating the culture case1103. In this case, the sealing glass1132is installed obliquely with respect to the optical axis X of the specimen observing portion1121so that the excitation light1142penetrating the culture case1103is reflected by the surface of the sealing glass1132and the reflected light will not return to the side of the living cells1129. The reflective member1122ais located with a normal at 45 degrees to the optical axis Y so that the excitation light1142penetrating the sealing glass1132is reflected perpendicularly to the optical axis Y. The light absorption member1122bis located on a reflected light path of the reflective member1122a. For example, a hair transplant paper1133is attached to the light absorption member1122bto absorb the excitation light.

In such a configuration, the first moving part1107is moved back and forth along the first guide1105by the first movement screw1106and the second moving part1111is moved up and down along the second guide1110by the second movement screw1108in order to set the height positions of the specimen observing portion1121and the dimmer means or dimmer unit1122relative to the respective culture case support racks1104. Moreover, the third movement screw1114and the fourth movement screw1116are driven so that the specimen observing portion1121and the dimmer means or dimmer unit1122move along the culture case support racks1104in order to position the specimen observing portion1121and the dimmer means or dimmer unit1122above the living cells1129in the culture case1103on the culture case support rack1104. In this case, the specimen observing portion1121and the dimmer means or dimmer unit1122are set in such a state that the optical axis X of the specimen observing portion1121coincides with the optical axis Y of the dimmer means or dimmer unit1122so that the living cells1129in the culture case1103are placed between them.

In this state, if the illumination light from the external illumination portion1123is introduced to the specimen observing portion1121through the incident-light fiber1124, the light projected from the incident-light fiber1124will be light converging on the rear focal plane of the objective lens1131through the first lens1134, and the light is selected by the excitation filter1125, reflected by the dichroic mirror1127and converges on the rear focal plane of the objective lens1131. The light converging on the rear focal plane of the objective lens1131is brought into parallel light by the objective lens1131to illuminate the illumination range uniformly as excitation light1142that excites the fluorescent dyes in the living cells1129in the culture case1103. The fluorescent dyes excited by the excitation light1142emit the fluorescence light1143having a wavelength longer than that of the excitation light1142. The fluorescence light1143is brought into the parallel light by the objective lens1131, penetrates the dichroic mirror1127and the emission filter1126, and falls on the image pickup plane of the image pickup device1128through the image-forming lens1144, thereby picking up an image thereof.

On the other hand, the excitation light1142that penetrates the culture case1103and that is not used for excitation reaches the dimmer means or dimmer unit1122and falls on the sealing glass1132. In this case, as the sealing glass1132is provided obliquely with respect to the optical axis X of the specimen observing portion1121, a slight amount of reflection on the surface of the sealing glass1132returns to the observed parts of the living cells1129without causing uneven illumination. Further, the excitation light1142that has penetrated the sealing glass1132is reflected by the reflective member1122a, reaches the light absorption member1122band is absorbed by the hair transplant paper1133.

Therefore, the excitation light1142that has penetrated the culture case1103and that is not used for excitation is led to and dimmed by the dimmer means or dimmer unit1122without returning to the side of the living cells1129, thereby making it possible to significantly reduce the discoloration of the fluorescent dyes in the living cells1129that are not targeted for observation. This allows the fluorescent image with good contrast to be observed for a long period of time.

Furthermore, since the hair transplant paper1133used for the dimmer means or dimmer unit1122is sealed in the dimmer means or dimmer unit1122by the sealing glass1132, it is also possible to prevent contamination inside the culture container1102.

Second Embodiment

Next, a second embodiment of the present invention will be described. The present embodiment is directed to another dimmer unit applicable instead of the dimmer unit of the first embodiment. Therefore, as the culture microscope apparatus to which the present embodiment is applied is similar to that described with reference toFIG. 1,FIG. 1is incorporated herein.

FIG. 3shows a schematic configuration of essential parts of the culture microscope apparatus of the present invention. InFIG. 3, the same numerals are given to the same parts as those inFIG. 2.

Dimmer means or a dimmer unit1201in the present embodiment has a second lens1202, a pin hole1203and a scattering portion1204. The pin hole1203is set greater than the diameter of the excitation light1142converged by the second lens1202. The scattering portion1204absorbs and scatters the incident excitation light1142and has low reflecting coating inside.

In such a configuration, the excitation light1142penetrating the culture case1103is converged into the pin hole1203by the second lens1202. The light that has passed through the pin hole1203enters the scattering portion1204. The scattering portion1204absorbs and scatters the incident excitation light1142with the low reflecting coating therein.

Thus, the excitation light1142is repeatedly absorbed and scattered in the scattering portion1204such that it is progressively dimmed to reduce energy. In this case, the excitation light repeatedly reaches the pin hole1203several times, thereby making it possible to sufficiently reduce damage to the living cells1129even if part of the light returns to the living cells1129.

Again, in this case, if the above-mentioned hair transplant paper1133is provided in the scattering portion1204and the sealing glass1132described in detail is provided in the pin hole1203, the degree of dimming can be increased while the contamination of the living cells1129is reduced.

Third Embodiment

Next, a third embodiment of the present invention will be described. The present embodiment is directed to another dimmer unit applicable instead of the dimmer unit of the first embodiment. Therefore, as the culture microscope apparatus to which the present embodiment is applied is similar to that described with reference toFIG. 1,FIG. 1is incorporated herein.

FIG. 4andFIG. 5show schematic configurations of essential parts of the culture microscope apparatus of the present invention. InFIG. 4andFIG. 5, the same numerals are given to the same parts as those inFIG. 2.

Dimmer means or a dimmer unit1301in the present embodiment has a third lens1302and a transmitted-light fiber1303, and further has an externally installed part1304attached to the culture microscope apparatus body1101. The third lens1302converges the excitation light1142that has penetrated the living cells1129to an end of the transmitted-light fiber1303. The transmitted-light fiber1303transmits the excitation light1142introduced through the third lens1302to the externally installed part1304.

In the externally installed part1304, a lens1302aand an illumination light source1305are located on an optical axis V at an end of the transmitted-light fiber1303, as shown inFIG. 5. A dimmer portion1306is located on an optical axis W vertical to the optical axis V at the end of the transmitted-light fiber1303. A reflecting mirror1307is located at an intersecting point Z of the optical axis V and the optical axis W. The reflecting mirror1307is located to form an angle of 45° to the optical axes V, W and is movable in a direction of the optical axis W.

The reflecting mirror1307is provided with a fifth guide1310and a fifth movement screw1311. The fifth guide1310has a fixed portion1310afixed to the side of the externally installed part1304and a moving portion1310b, and allows the moving portion1310bto move linearly relative to the fixed portion1310a. Further, the fifth movement screw1311has a screw portion1311afixed, in a manner to be able to turn, to the side of the externally installed part1304and a nut portion1311b, and the screw portion1311ais turned so that the nut portion1311bmoves straight.

The reflecting mirror1307is attached to the moving portion1310bof the fifth guide1310and the nut portion1311bof the fifth movement screw1311. Further, a fifth drive portion1312is attached to the screw portion1311aof the fifth movement screw1311. The fifth drive portion1312rotationally drives the screw portion1311aof the fifth movement screw1311under instructions from the controller1138, and the rotation of the screw portion1311ais converted to straight movement of the nut portion1311b, thereby enabling the reflecting mirror1307to move in a direction (direction of the optical axis W) guided by the fifth guide1310.

Position sensors1308a,1308bare located on a travel path of the reflecting mirror1307. The position sensor1308adetects that the reflecting mirror1307is located at the intersecting point Z of the optical axis V and the optical axis W, and the position sensor1308bdetects that the reflecting mirror1307is located, on the optical axis W, at an evacuated position where it does not prevent the excitation light projected from the transmitted-light fiber1303. Thereby, the controller1138can control the position of the reflecting mirror1307by use of the detected positions of the position sensors1308a,1308b, so that an optical connection end of the transmitted-light fiber1303can be switched between the dimmer portion1306and the illumination light source1305.

According to such a configuration, when the reflecting mirror1307is located at the intersecting point Z, the excitation light1142projected from the transmitted-light fiber1303is brought into parallel light by the lens1302a, and is reflected by the reflecting mirror1307and reaches the dimmer portion1306. In this case, again, if the above-mentioned hair transplant paper1133is provided in the dimmer portion1306, the excitation light1142is absorbed and dimmed by the hair transplant paper1133.

On the other hand, when the reflecting mirror1307is moved to the evacuated position on the optical axis W where it does not prevent the excitation light projected from the transmitted-light fiber1303, the transmitted-light fiber1303is optically connected to the illumination light source1305, so that the light from the illumination light source1305is projected from the third lens1302through the transmitted-light fiber1303, and is applied to the living cells1129from the opposite side of the specimen observing portion1121with reference to the living cells1129.

Thus, the position of the reflecting mirror1307can be switched to lead the excitation light1142that has penetrated the culture case1103to the side of the dimmer portion1306for dimming in the case of incident-light illumination so that the discoloration of the living cells1129can be prevented. On the one hand, if the light from the illumination light source1305illuminates the living cells1129at the time of transmitted-light illumination, a transmitted image of the living cells1129can be observed.

Fourth Embodiment

Next, a fourth embodiment of the present invention will be described. The present embodiment is directed to another dimmer unit applicable instead of the dimmer unit of the first embodiment. Therefore, as the culture microscope apparatus to which the present embodiment is applied is similar to that described with reference toFIG. 1,FIG. 1is incorporated herein.

FIG. 6shows a schematic configuration of essential parts of the culture microscope apparatus of the present invention. InFIG. 6, the same numerals are given to the same parts as those inFIG. 3.

The culture microscope apparatus of the present embodiment comprises a transmitted-light fiber1401, a fourth lens1402and an externally provided illumination light source1403, in addition to the dimmer means or dimmer unit1201described in the second embodiment. The illumination light source1403is optically connected to the fourth lens1402through the transmitted-light fiber1401, and light from the illumination light source1403is projected from the fourth lens1402through the transmitted-light fiber1401.

According to such a configuration, if the optical axis Y of the dimmer means or dimmer unit1201is adapted to coincide with the optical axis X of the specimen observing portion1121, the excitation light1142that has penetrated the culture case1103is introduced into the dimmer means or dimmer unit1201and dimmed.

Furthermore, if the dimmer means or dimmer unit1201is moved by the fourth guide1115, the fourth movement screw1116and the fourth drive portion1118, and an optical axis T on the side of the fourth lens1402and the transmitted-light fiber1401is adapted to coincide with the optical axis X of the specimen observing portion1121, the light from the illumination light source1403is projected from the fourth lens1402through the transmitted-light fiber1401, and is applied to the living cells1129from the opposite side of the specimen observing portion1121with reference to the living cells1129. This enables the living cells1129to be illuminated with the light from the illumination light source1403to obtain a transmission observation image.

In this way, the excitation light1142can be dimmed and observation of the living cells1129with transmitted illumination can be performed without specially providing means or a mechanism to switch the position of the reflecting mirror1307described in the third embodiment.

Fifth Embodiment

The background of the present embodiment will first be described.

Because living organisms have a high degree of complexity, it is not easy to understand their structures and functions. Therefore, a simple experiment system has recently been utilized, and it uses cells that are minimum units capable of reproducing a life phenomenon, that is, the cultured cells. The cultured cells are used to enable an experiment in which analyses of a hormone response and the like are not affected by other factors in the living organism.

In other words, functional analysis of genes can be implemented by the introduction and inhibition of genes. It is necessary to use an environment simulating the inside of the living organism in order to culture cells. Therefore, the temperature is set at a body temperature of 37° C., and a culture medium simulating intercellular fluid is used. The culture medium includes a carbonic acid buffer for PH adjustment in addition to sources of nutrition such as amino acid. The carbonic acid buffer is in an equilibrium state in the presence of air containing carbon dioxide gas at a high partial pressure of 5%, and used for culture in an open system such as a dish. Moreover, a highly humid environment is required to prevent water from evaporating from the culture medium.

To culture cells, a carbon dioxide gas incubator that has the above-mentioned environmental conditions is used.

A phase contrast microscope is used to observe the state of cells and a fluorescence microscope is used to observe the expression of GFP, thereby performing time-lapse observation based on time-series image acquirement.

However, the microscope is generally placed outside the carbon dioxide gas incubator, the observation causes changes in, for example, temperature and PH to cells to give stress to the cells, which might affect an experimental result.

Therefore, Jpn. Pat. Appln. KOKAI Publication No. 2003-93041 proposes an apparatus in which a movable device and a microscope are arranged in an incubator capable of controlling carbon dioxide gas, temperature and humidity. This apparatus makes it possible to observe cells in a culture environment without taking the cultured cells from the culture environment.

Furthermore, Jpn. Pat. Appln. KOKAI Publication No. 10-28576 proposes a microscopic observation transparent constant temperature culture apparatus that is installed on a microscope and that can control carbon dioxide gas, temperature and humidity.

If the cultured cells are contaminated with microorganisms such as bacteria and molds having a high proliferation rate, the cultured cells run out of nutrients and die. Culture instruments are disinfected and sterilized for prevention of contamination, but the apparatus of Jpn. Pat. Appln. KOKAI Publication No. 2003-93041 comprises moving means or a moving mechanism in the culture apparatus, thus having a complicated shape in the apparatus and having difficulty in cleaning. This will be a factor that restricts the sufficient prevention of contamination.

Moreover, in the apparatus of Jpn. Pat. Appln. KOKAI Publication No. 10-28576, a container is constructed in a limited space on the microscope, so that temperature setting is easily changed by the atmospheric environment.

In view of such circumstances, the present embodiment is directed to the culture microscope apparatus that causes a small change in an installed environment and that is easily cleaned.

FIG. 8schematically shows the culture microscope apparatus of a fifth embodiment of the present invention. The culture microscope apparatus basically comprises a microscope device to observe cultured cells and a culture device2101capable of controlling temperature, humidity and carbon dioxide gas concentration so that they have values suitable for the cultured cells.

InFIG. 8, the microscope device comprises an objective lens2125, which allows of observation of the cultured cells in a specimen2123(i.e., culture case containing the cultured cells), an image pickup device2150, which picks up an image of the cultured cells enlarged by the objective lens2125, a moving device, which relatively moves the specimen2123(i.e., the cultured cells) and the objective lens2125, an upper base member2114on which the objective lens2125and the moving device are placed, a lower base member2191on which the image pickup device2150is placed, and support columns2111coupling the upper base member2114to the lower base member2191.

The upper base member2114, the lower base member2191and the support columns2111are all made of a low expansion material with a little expansion due to heat.

The moving device comprises a straight moving guide2115, a horizontally moving member2116, a ball screw2117, a stepping motor2118, a rotation shaft2119, a rotation shaft bearing2120, a stepping motor2121, a table2122, a straight moving guide2124, a vertically moving member2126, a ball screw2127and a stepping motor2128.

The straight moving guide2115, the horizontally moving member2116, the ball screw2117and the stepping motor2118are all provided under the upper base member2114. The straight moving guide2115supports the horizontally moving member2116movably in one direction, and the stepping motor2118moves the horizontally moving member2116through the ball screw2117. The horizontally moving member2116holds the rotation shaft bearing2120and the stepping motor2121. The stepping motor2121is located coaxially with the rotation shaft bearing2120. The rotation shaft bearing2120supports the rotation shaft2119rotatably vertically to a horizontal plane and pressurizes the rotation shaft2119. Balls2135are provided between the rotation shaft2119and the rotation shaft bearing2120to reduce friction (seeFIG. 9). A lower end of the rotation shaft2119is connected to the stepping motor2121. An upper end of the rotation shaft2119protrudes from an upper surface of the upper base member2114through an opening formed in the upper base member2114. The table2122on which the specimen2123is mounted is fixed detachably by screws to the upper end of the rotation shaft2119.

The objective lens2125is located on a straight line extending from the rotation shaft2119in a moving direction of the horizontally moving member2116. The objective lens2125is supported by the vertically moving member2126. The vertically moving member2126is supported so that it can be moved vertically by the straight moving guide2124fixed to the upper base member2114. Further, the vertically moving member2126is coupled to, through the ball screw2127, the stepping motor2128fixed to the upper base member2114, and moved vertically by the stepping motor2128. The straight moving guide2124, the vertically moving member2126, the ball screw2127and the stepping motor2128are all provided under the upper base member2114.

The moving device thus configured enables the relative movement of the specimen2123mounted on the table2122and the objective lens2125. That is, the horizontally moving member2116can move linearly in one direction in the horizontal plane relative to the upper base member2114. Further, the table2122can make rocking movement with respect to the horizontally moving member2116. Moreover, the vertically moving member2126can move linearly in a vertical direction with respect to the upper base member2114. That is, the specimen2123can move linearly in one direction and rock with respect to the objective lens2125, and the objective lens2125can move relatively in a vertical direction with respect to the specimen2123. As a result, the specimens2123can be observed. Moreover, because the image pickup device2150is located outside the culture device2101, noise resulting from the temperature of the image pickup device2150can be reduced. Further, the upper base member2114and the lower base member2191that have different temperatures inside and outside the culture device2101are made of the low expansion material, such that distortion due to thermal expansion can be reduced and the adjustment of the optical system is not disturbed.

[Outline of Culture Device]

The culture device2101comprises the upper base member2114, a door2101alocated above the upper base member2114, and a base portion2101blocated under the upper base member2114. The door2101acan open and close with respect to the upper base member2114to mount the specimen2123on the table2122. When the door2101ais closed, the upper base member2114and the door2101adefine a culture space. In order to keep an airtight state between the door2101aand the upper base member2114when the door2101ais closed, an elastic seal member2112is provided between the door2101aand the upper base member2114. The base portion2101bis held by the support columns2111, and the elastic seal member2112is provided between the base portion2101band the upper base member2114to keep an airtight state between the base portion2101band the upper base member2114.

The culture device2101comprises an insulating material2102, a metallic interior portion2103made of stainless steel having antibacterial and corrosion resistance properties or anti-bacterially coated, a sensor2104to sense the temperature, humidity and PH in the culture device, a heater2105provided in the interior of the culture device for internal temperature adjustment, humidification means or a humidification heater2106provided on the upper base member2114for temperature adjustment in the culture device, an electromagnetic valve2107that is connected to a carbon dioxide gas cylinder to adjust the carbon dioxide gas concentration for PH adjustment and that supplies carbon dioxide gas, a column heater2108to adjust the temperature of the support columns2111coupling the inside and outside of the culture device2101, and a support column sensor2109to measure the temperature of the support columns2111. On the upper base member2114, a humidification pad2113containing water for humidification in the culture device2101is placed at a position above the humidification heater2106. The culture device2101further comprises a controller2110to perform an operation for maintenance of a set condition in accordance with a signal from the sensor2104in order to control the heater2105, the humidification heater2106and the electromagnetic valve2107.

According to such a configuration, the insulating material2102thermally isolates the image pickup device2150from the culture device2101. The insulating material2102and the seal member2112reduce heat going in and out of the culture device2101. This reduces the influence of outdoor air temperature changes and enables the stabilization of temperature. Moreover, as the less heat goes in and out, the capacity of the heater2105and the humidification heater2106and water capacity of the humidification pad2113can be reduced. Further, as plenty of nutrients are provided to the culture medium where the cells are cultured, microorganisms having high reproduction ability infiltrating therein cause an adverse effect to the cultured cells. However, the interior portion2103of the culture device2101having the antibacterial and corrosion resistance properties prevents the infiltration of the microorganisms and can maintain the activity of the cultured cells.

Furthermore, high humidity is maintained in the culture space of the culture device2101to prevent the culture medium from being dried due to the evaporation of water. Therefore, if the specimen operated at a room temperature of about 23° C. is brought into the culture device, condensation is caused on the outside of the container, resulting in the deterioration of the observation image through the microscope. It is thus preferable that humidification control described below is performed for the culture device2101. The culture device2101has a door sensor, which is not specifically shown, to sense that the door2101ais opened or closed. In accordance with a signal (an instruction to mount the specimen2123) from the door sensor, the controller2110causes the humidification heater2106to warm the humidification pad2113to start humidification after a certain period of time has passed (e.g., after ten minutes) so that the cultured cells including the container (i.e., the specimen2123) will be at the same temperature as that inside the culture device2101. Owing to this control, the cultured cells are humidified after reaching the same temperature as that inside the culture device2101, whereby a satisfactory microscope observation image can be obtained without causing condensation in the cultured cells including the container. Moreover, if a specimen mounting button is provided in the controller2110instead of the door sensor and the controller2110performs similar humidification control in accordance with an instruction based on the specimen mounting button, similar effects can be provided. In addition, instead of turning the humidification heater2106on and off, a door provided for the humidification pad2113may be opened and closed.

[Details of Table Section and Objective Lens Section of Microscope Device]

FIG. 9shows in an enlarged manner a peripheral part of the table shown inFIG. 8. Between the table2122and the upper base member2114, an intermediate member2129is located fixedly to the horizontally moving member2116. Between the table2122and the intermediate member2129, there are provided two ring-shaped seat seals2199made of ethylene tetrafluoride (PFTE), and an elastic O-ring2130. The O-ring2130is located in a pressed state. Also, between the upper base member2114and the intermediate member2129, there are provided the two seat seals2199and the O-ring2130, and the O-ring2130is located in a pressed state.

FIG. 10shows in an enlarged manner a peripheral part of the objective lens shown inFIG. 8. The upper base member2114has an opening2114bto protrude the objective lens2125, and a groove is formed on an inner peripheral surface of the opening2114b, and an elastic O-ring2134is received in the groove of the opening2114b. In such a state that the objective lens2125protrudes out of an upper surface of the upper base member2114through the opening2114b, the O-ring2134is pressed.

InFIG. 9, the rotation of the stepping motor2121rocks the table2122. The table2122rocks such that sliding is mainly caused between the two seat seals2199of the PFTE material having a low friction coefficient. Even if a distance change is caused between the table2122and the intermediate member2129through the seat seals2199, the O-ring2130elastically deforms to absorb the distance change to always prevent gap formation. Similarly, even if a distance change is caused between the upper base member2114and the intermediate member2129through the seat seals2199, the O-ring2130works similarly to prevent the gap formation. Moreover, as the seat seals2199have a low friction coefficient, sliding resistance is kept low. Even if members constituting the guides and stages have lower rigidity, accurate movement is possible. Further, low frictional force can prevent the abrasion of the members.

Furthermore, inFIG. 10, a distance change is also caused between the objective lens2125and the opening2114bof the upper base member2114in the vertical movement of the objective lens2125, but the O-ring2134elastically deforms to absorb the distance change to always prevent gap formation.

InFIG. 8, if the door2101ais closed, the seal member2112is pressed between the door2101aand the upper base member2114. Therefore, no gap is formed between the door2101a, that is, the culture device2101and the upper base member2114.

Thus, the microscope device is isolated from the moisture of the culture device2101by the upper base member2114, the base portion2101band sealing structures (the seal member2112, the seat seals2199, the O-ring2130and the O-ring2134). In other words, the upper base member2114, the base portion2101band the sealing structures (the seal member2112, the seat seals2199, the O-ring2130and the O-ring2134) constitute isolation means or an isolator to isolate the microscope device from the moisture of the culture device2101.

This configuration can keep the culture space defined by the closed door2101aand the upper base member2114from the outside air. Further, the table2122and the objective lens2125inside the culture space can be moved from the outside of the culture space. It is thus easy to maintain the temperature and humidity of the culture space, so that necessary heater capacity and a necessary amount of water can be reduced. It is further possible to reduce the infiltration of moisture into a mechanism part (the moving device) and an optical part, enabling the prevention of rust and condensation.

Furthermore, the use of PTFE having a low friction coefficient for a sealing surface enables a microscope having both positional accuracy and sealing performance.

Even with the above-described sealing structures (the seal member2112, the seat seals2199, the O-ring2130and the O-ring2134), moisture slightly infiltrates into a lower surface side of the upper base member2114. In order not to expose the mechanism part (the moving device) and the optical part to such moisture, inFIG. 9, the gap between the lower surface of the upper base member2114and the horizontally moving member2116is set at 0.1 mm or less, and an intake pipe2131and an exhaust pipe2132are connected to the horizontally moving member2116. The intake pipe2131and the exhaust pipe2132are led to the external, and an air pressure source is connected to the intake pipe2131for air intake. The intake pipe2131is wound several times in the culture device2101to elongate a passage in the culture device2101. The intake pipe2131and the exhaust pipe2132constitute dehumidifying means or a dehumidifier that connects the atmosphere in the microscope device to the air outside the culture microscope apparatus and that dehumidifies the atmosphere in the microscope device. Further, the intermediate member2129comprises, at a cylindrical portion connected to the horizontally moving member2116, a communication hole2129apenetrating the cylindrical portion.

InFIG. 10, the upper base member2114is provided with a capturing member2133extending from the upper base member2114in the axial direction of the objective lens2125. The capturing member2133has an opening through which the objective lens2125passes, and the O-ring2134is mounted in the groove formed in the inner peripheral surface of the opening to press the objective lens2125. The above-mentioned intake pipe2131and the exhaust pipe2132are also connected between the O-ring2134of the capturing member2133and the O-ring2134of the upper base member2114.

The moisture that has infiltrated from the culture space defined by the closed door2101aand the upper base member2114through the space between the two seat seals2199diffuses between the upper base member2114and the horizontally moving member2116from a gap between the intermediate member2129and the rotation shaft2119through the communication hole2129a. The moisture diffused between the upper base member2114and the horizontally moving member2116is discharged to the outside from the exhaust pipe2132together with the outside air introduced from the intake pipe2131. The outside air introduced from the intake pipe is warmed by the long passage in the culture device2101and reaches the horizontally moving member2116without dropping the temperature of the members. The moisture that has infiltrated from the space between the objective lens2125and the O-ring2134is similarly discharged to the outside.

The configuration described above also forces the slight amount of moisture that has infiltrated from each of the sealing structures (the seat seals2199, the O-ring2130and the O-ring2134) to be discharged to the outside. Thus, moisture does not reach the mechanism part (the moving device) and the optical part without the fear of rust and condensation. Further, a constant temperature of the objective lens2125can eliminate focal movement of the objective lens2125due to temperature changes. This enables a long-term observation without defocusing.

Next, a manipulator to introduce genes and drugs into the cultured cells will be described referring toFIG. 10. The manipulator has an arm2142capable of rocking and vertical movement, and the arm2142holds a syringe2143at an end. The arm2142is detachably fixed to a vertically moving shaft2138by screws. The vertically moving shaft2138is received in a cylindrical rotation shaft2136and can move vertically with respect to the rotation shaft2136. The rotation shaft2136is attached to the upper base member2114through a bearing2137and can rotate with respect to the upper base member2114. The rotation shaft2136comprises a main wheel2195at a lower end. The main wheel2195engages with a pinion2193attached to an output shaft of a stepping motor2194fixed to the upper base member2114. The vertically moving shaft2138has a female screw at a lower end. The female screw of the vertically moving shaft2138engages with a male screw formed in the output shaft of a stepping motor2140. The vertically moving shaft2138has a groove2138aextending vertically in an outer peripheral surface. A pin2139fixed to the rotation shaft2136is received in the groove2138a. The pin2139determines the range of vertical movement of the vertically moving shaft2138with respect to the rotation shaft2136, and regulates the rotation of the vertically moving shaft2138with respect to the rotation shaft2136. A position to fix the arm2142to the vertically moving shaft2138is adjusted so that the distance between the syringe2143and a core of the rotation shaft2136will be the same as the distance between an optical axis of the objective lens2125and the core of the rotation shaft2136.

In the configuration described above, the arm2142can be moved to locate the syringe2143at the cells in the center of a viewing field even in a highly humid environment, and for example, a reagent placed on the upper base member2114can be administered to the cultured cells. Further, the stepping motor2194to rotate the arm2142and the stepping motor2118to horizontally move the specimen2123can be driven in conjunction with each other to locate the syringe2143at an optional position in a predetermined range.

In the culture microscope apparatus of the present embodiment, when the table2122and the arm2142are removed, the only members protruding on the upper side of the upper base member2114are cylindrical members (the rotation shaft2119and the intermediate member2129of the moving device, the rotation shaft2136of the manipulator), so that cleaning can be easily performed.

A water supply device for a water-immersion objective lens will be described referring toFIG. 10. The culture microscope apparatus of the present embodiment comprises the water supply device, which supplies water to the water-immersion objective lens, considering the case where the objective lens2125is a water-immersion objective lens. The water supply device comprises a cooler capable of setting a temperature different from a set temperature of the culture device2101. The cooler comprises, but not limited to, a peltier element2144in the present embodiment. On a lower surface of the table2122, the peltier element2144is fixed at a place where it can be located on the optical axis of the objective lens2125when the table2122moves. The peltier element2144has a water supply surface2144athat can face the objective lens2125. The culture space is maintained in a saturated state close to a relative humidity of 100%, so that if the water supply surface2144aof the peltier element2144is cooled off several times, steam is condensed on the water supply surface2144a. The table2122is moved by the above-mentioned moving device to locate the water supply surface2144aabove the objective lens2125and then the table2122is lowered such that water condensed on the water supply surface2144acan be supplied to the objective lens2125. According to this configuration, water can be supplied to the objective lens2125only by the peltier element2144without using an extra member. Thus, an inexpensive water supply device can be provided.

The peltier element2144can be provided not on the table2122but on the upper surface of the upper base member2114, and the above-mentioned manipulator can be used to supply water condensed on the upper surface of the peltier element2144to the objective lens2125. This configuration can reduce the temperature changes of the specimen because the peltier element2144is not provided on the table2122.

The microscope device is capable of fluorescent observation and dark field observation. The fluorescent observation is used to identify the expression of fluorescent protein at a target part, and the dark field observation is used to visualize the nucleus and outline of cells for confirmation of the positions of cells, the state of culture or bacterial contamination.

The microscope device includes an illumination device for illuminating the cultured cells and an observation device to observe the cultured cells.FIG. 11shows the illumination device applicable to the microscope device shown inFIG. 8. As shown inFIG. 11, the illumination device comprises light emitting diodes2145having different emission wavelengths, excitation filters2146located in front of the light emitting diodes2145, and a bending member2147. The light emitting diodes2145, the excitation filters2146and the bending member2147are all located at an outer peripheral part of the objective lens2125. The excitation filters2146selectively transmit light having a specific wavelength among wavelengths of illumination light emitted from the light emitting diodes2145. The bending member2147bends the illumination light that has penetrated the excitation filters2146and orients it toward the specimen2123.

As shown inFIG. 8, the observation device comprises the objective lens2125, an image-forming lens2149, which cooperates with the objective lens2125to constitute an image-forming optical system, the image pickup device2150, which picks up an optical image formed by the image-forming optical system, and a monitor2192to display the image obtained by the image pickup device2150. The base portion2101bis provided with an optical window2223so that light from the specimen2123travels to the image pickup device2150through the objective lens2125and the image-forming lens2149. The optical window2223may comprise a transparent optical member such as glass plate. The observation device further comprises an emission filter2148, which selectively transmits light having a specific wavelength among wavelengths of observation light directed to the image pickup device2150, and a turret2151to locate the emission filter2148on the optical axis as required. The image pickup device2150is preferably a cooled CCD considering the fluorescent observation.

In the dark field observation, inFIG. 8, the turret2151is switched to locate an air hole on the optical axis and displace the emission filter2148from the optical axis. InFIG. 11, the light emitted by the light emitting diodes2145penetrates the excitation filters2146, and illuminates the specimen2123due to the bending member2147from the outside of NA of the objective lens2125. Therefore, the illumination light and the light regularly reflected by the lower surface of the specimen container are not captured by the objective lens2125. The reflected light and scattered light alone due to the cultured cells in the specimen2123are captured by the objective lens2125and detected by the image pickup device2150. Thus, even transparent cultured cells can be visualized without being dyed. Further, because a generally used transmitted-light illumination portion used in phase difference observation is not necessary, a space is produced above the specimen, thus facilitating operations including taking the specimen2123in and out of the culture device2101and administering the reagent to the specimen2123.

FIG. 12shows another illumination device applicable to the microscope device shown inFIG. 8. If NA of the objective lens2125is, for example, 0.85, the range of light captured by the objective lens2125will be about 60 degrees from the optical axis. Illumination optical axes are set on a bus at 70 degrees from the optical axis toward the center of a specimen container bottom surface, and an excitation filter2152, a collimating lens2153and a light emitting diode2154are located on the illumination optical axes. The excitation filter2152, the collimating lens2153and the light emitting diode2154are sealed by a dustproof glass2156inside an illumination member2155provided out of a moving range of the table2122on the upper base member2114.

In other words, the illumination device ofFIG. 12comprises the light emitting diode2154to emit the illumination light, the collimating lens2153to form the illumination light emitted by the light emitting diode2154into parallel light, the excitation filter2152, which selectively transmits light having a specific wavelength among wavelengths of the illumination light emitted from the light emitting diode2154, the illumination member2155housing the light emitting diode2154, the collimating lens2153and the excitation filter2152, and the dustproof glass2156constituting an optical window provided in the illumination member2155. The illumination optical axis passing the light emitting diode2154, the collimating lens2153and the excitation filter2152is inclined at 70 degrees with respect to the optical axis of the objective lens2125.

The light emitted by the light emitting diode2154is brought into parallel light by the collimating lens2153, and illuminates uniformly within an observation field in the objective lens2125. The light that has penetrated the specimen2123is the light outside the NA of the objective lens2125, and is therefore not captured by the objective lens2125. The reflected light and scattered light alone due to the specimen2123are captured by the objective lens2125and detected by the image pickup device2150. Therefore, effects similar to those in dark field illumination by the illumination device shown inFIG. 11are provided; for example, even transparent cultured cells can be visualized without being dyed, and the transmitted-light illumination portion is not necessary. When the fluorescent observation is not performed together or when narrow-band wavelengths are not necessary in an excitation wavelength, the excitation filter2152may be removed from the configuration.

The fluorescent observation can also be performed using the illumination device inFIG. 11. In the fluorescent observation, inFIG. 8, the turret2151is switched to locate on the optical axis the emission filter2148adapted to a fluorescent wavelength of the specimen2123. InFIG. 11, light having a wavelength that is needed to excite the specimen among wavelengths of light emitted from the light emitting diode2154is selectively transmitted by the excitation filter2146and illuminates the specimen2123. The specimen2123excited by the illumination light emits fluorescence light having a wavelength longer than the wavelength used for the excitation. The fluorescence light is captured by the objective lens2125, brought into parallel light and exits from the objective lens2125to reach the emission filter2148. The light that has penetrated the emission filter2148is imaged on a light receiving surface of the image pickup device2150by the image-forming lens2149, and displays an object image on the monitor2192. Owing to the dark field observation, the illumination light is not captured by the objective lens2125, so that SN in accordance with auto-fluorescence in the objective lens2125is not reduced by the illumination light.

The fluorescent observation can also be performed using the illumination device inFIG. 12. InFIG. 12, the light emitted by the light emitting diode2154is brought into parallel light by the collimating lens2153, and the light needed to excite the specimen is selectively transmitted by the excitation filter2152and illuminates the specimen uniformly. The function leading to the image-forming is the same as in the illumination device inFIG. 11.

Furthermore, instead of the light emitting diode2154, a light source provided outside the apparatus may be used in such a manner to transmit through a fiber. When the fiber is used, a high luminance light source can be used because it is not necessary to consider the intensity of light and heat of the light source.

Here, the illumination device for the fluorescent observation is an oblique illumination device as shown inFIG. 11andFIG. 12, but it may also be an incident-light illumination device. That is, the illumination device for the fluorescent observation includes the objective lens2125and may have the configuration similar to those of the illumination devices described in the first embodiment to fourth embodiment.

FIG. 13shows a modification of the culture microscope apparatus of the present embodiment. More specifically, the culture microscope apparatus that has changed the microscope device shown inFIG. 8from the configuration suitable for the dark field observation to the configuration suitable for the phase difference observation is shown. InFIG. 13, members indicated by the same numerals as the members shown inFIG. 8are the same and will not be described in detail.

In the culture microscope apparatus of the present modification, the microscope device comprises a phase difference objective lens2157in place of the objective lens2125inFIG. 8and also comprises a transmitted-light illumination device in place of the dark field illumination device inFIG. 11andFIG. 12, as understood fromFIG. 13.

The transmitted-light illumination device comprises an illumination support column2158, a ring slit2159, a light emitting diode2160, a reflecting mirror2161and a collimating lens2162. The illumination support column2158is placed out of the moving range of the table2122on the upper base member2114. The illumination support column2158houses the ring slit2159, the light emitting diode2160and the reflecting mirror2161. The collimating lens2162is inserted into an opening of the illumination support column2158and closely fixed by an adhesive material. The ring slit2159has a ring-shaped opening and is located at a position conjugate with a rear focal plane of the phase difference objective lens2157. The light emitting diode2160is located in the vicinity of the ring slit2159.

The phase difference objective lens2157comprises a phase plate2163on the rear focal plane. The size of the phase plate2163includes the projected ring slit2159. That is, an image of the ring slit2159is projected on an inner side of the phase plate2163. Moreover, the phase plate2163comprises an optical member that shifts the phase of transmitted light by ¼ wavelengths, and an ND film that attenuates the transmitted light.

Light emitted from the light emitting diode2160passes through the opening of the ring slit2159, has its direction changed by the reflecting mirror2161, is brought into parallel light by the collimating lens2162, and illuminates the specimen2123uniformly.

Zero-th light that has penetrated the specimen2123converges on the phase plate2163of the phase difference objective lens2157, and is subjected to phase shift and light amount attenuation. Further, primary light diffracted at the specimen2123does not converge on the phase plate2163on the rear focal plane of the phase difference objective lens2157, and is not therefore subjected to phase shift and light amount attenuation.

The zero-th light and the primary light are imaged on a light receiving surface of the image pickup device2150by the image-forming lens2149. The phase shift of the zero-th light performed by the phase plate2163causes interference between the zero-th light and the primary light, so that even undyed specimens can be observed. Moreover, the collimating lens2162shuts off the culture space from the inside of the illumination support column2158, so that condensation is not caused on optical members inside the illumination support column.

<Another Modification of Fifth Embodiment>

FIG. 15shows another modification of the culture microscope apparatus of the fifth embodiment of the present invention. More specifically, the culture microscope apparatus is shown in which a dimmer unit is added to the culture microscope apparatus shown inFIG. 8. InFIG. 15, members indicated by the same numerals as the members shown inFIG. 8are the same and will not be described in detail.

In the culture microscope apparatus of the present modification, a dimmer unit2188is located above the objective lens2125. The dimmer unit2188faces the objective lens2125, and the specimen2123to be observed is properly located between the dimmer unit2188and the objective lens2125. The dimmer unit2188is supported by a support column2189, and the support column2189is fixed to the upper base member2114out of the moving range of the table2122. The dimmer unit2188may comprise the dimmer unit described in the first embodiment to fourth embodiment. That is, the dimmer unit2188has the same configuration as those of the dimmer unit1122, the dimmer unit1201or the dimmer unit1301.

According to the present modification, it is possible to significantly reduce the discoloration of the fluorescent dyes that are not targeted for observation, and to observe the fluorescent image with good contrast for a long period of time.

<Still Another Modification of Fifth Embodiment>

FIG. 15shows still another modification of the culture microscope apparatus of the fifth embodiment of the present invention. InFIG. 15, members indicated by the same numerals as the members shown inFIG. 8are the same and will not be described in detail.

The upper base member2114in the culture device2101is set up with a leg attachment portion2202fixed through support columns2201, and the center of the leg attachment portion2202and the lower base member2191are connected by a leg portion2205.

Furthermore, the following member is used as a seal member2203of the objective lens2125. The seal member2203is a thin elastic rubber material, and has a cylindrical portion and two planar portions. The two planar portions are fixed to the objective lens2125and the upper base member2114through a fixing member2204.

The leg attachment portion2202and the lower base member2191that have different temperature settings inside and outside the culture device2101are connected by the leg portion2205, so that the distortion of the material caused by an expansion difference due to the temperature can be reduced and the optical adjustment is not disturbed.

Furthermore, the seal member2203enables sealing with a low sliding resistance, allowing an improvement in positional repeatability.

<Further Still Another Modification of Fifth Embodiment>

FIG. 16shows further still another modification of the culture microscope apparatus of the fifth embodiment of the present invention. InFIG. 16, members indicated by the same numerals as the members shown inFIG. 8are the same and will not be described in detail.

The culture microscope apparatus of the present modification further comprises a transmitted-light illumination device2210for transmitted-light illumination of the specimen2123, in addition to the construction of the culture microscope apparatus shown inFIG. 8. The transmitted-light illumination device2210, which is located above the objective lens2125, is attached to the door2101aso as to be detached from the door2101a. The door2101ais provided with two optical windows2221and2222, which transmit light from the transmitted-light illumination device2210. The optical windows2221and2222may comprise transparent optical members such as glass plates.

The culture microscope apparatus has a region I that is basically defined by the door2101aand the upper base member2114, a region II that is basically defined by the base portion2101band the upper base member2114, and a region III under the culture device2101. In the region I, the temperature and humidity are maintained at a level equal to that in an environment inside a body of a human being, an animal or a plant. In the region II, the temperature is on a level equal that of the region I and the humidity is maintained at a normal humidity level. In the region III, the temperature and humidity are on a normal temperature and normal humidity level.

The regions I, II and III are located along the optical axis of the image-forming optical system including the objective lens2125, the image-forming lens2149and the image pickup device2150. The specimen2123is located in the region I, and the image pickup device2150in the region III.

The regions II and III are optically connected through the optical window2223, which is positioned between the regions II and III, so that light for the image-forming optical system, light from the specimen2123, passes through a boundary between the regions II and III. The region I is optically connected to the transmitted-light illumination device2210through the optical windows2221and2222, which are positioned between the region I and the transmitted-light illumination device2210, so that light for the image-forming optical system, light from the transmitted-light illumination device2210, passes into the region I.

The regions I and II satisfy the following conditional equations:
36° C.≦t1≦38° C., 90%≦h1≦100%
36° C.≦t2≦38° C., 50%≦h2≦80%
where t1indicates the temperature of the region I, h1indicates the humidity of the region I, t2indicates the temperature of the region II, and h2indicates the humidity of the region II.

The specimen2123and the objective lens2125, which are weak in heat retaining and moisture retaining performance, are preferably separated from the normal temperature and normal humidity environment. An adverse effect (especially condensation) on the objective lens2125due to the culture environment is desirably reduced as much as possible. The image pickup device2150in the image-forming optical system, which is basically heating elements, is desirably located as far away from the culture environment as possible.

The culture microscope apparatus of the present modification has the region II as a buffer region, through which the specimen2123and the image pickup device2150are positioned and in which the objective lens2125is mainly located, so that the adverse effect (especially condensation) on the objective lens2125is effectively reduced and the culture environment is easily maintained in preferable condition.

Sixth Embodiment

The present embodiment is also directed to the culture microscope apparatus that causes a small change in the installed environment and that is easily cleaned, similarly to the fifth embodiment.

FIG. 17schematically shows the culture microscope apparatus of a sixth embodiment of the present invention.

The culture microscope apparatus of the present embodiment comprises an incubator2164as a culture device, which can control temperature, humidity and PH and is used for the cultured cells, a microscope device2165housed in the incubator2164, and a slide device2166that moves the microscope device2165between the inside and outside of the incubator2164. The slide device2166is provided in the incubator2164.

The microscope device2165comprises an illumination device2167for illuminating the cultured cells, an observation device2168to observe the cultured cells, a moving device2169, and a microscope container2170. The illumination device2167comprises a light emitting diode2171, a collimating lens2172, and an excitation filter2173. The observation device2168comprises an objective lens2174, a turret2176equipped with an emission filter2175, an image-forming lens2177, and an image pickup device2178. The objective lens2174and the image pickup device2178constitute an image-forming optical system. The moving device relatively moves the specimen and the objective lens2174placed in the microscope container2170, and comprises a horizontal stage2179capable of two-dimensional movement in a horizontal plane and a vertical stage2180placed on the horizontal stage2179and capable of vertical movement.

The objective lens2174and the illumination device2167are integrally fixed to the vertical stage2180. The emission filter2175, the turret2176, the image-forming lens2177and the image pickup device2178are integrally fixed to the horizontal stage. The microscope container2170is substantially a rectangular-parallelepiped-shaped box having two openings. One opening is located on an upper surface of the microscope container2170, while the other opening is located on a side surface of the microscope container2170. An elastic O-ring2181is placed in a groove provided in the vicinity of the opening on the upper surface, and an optically transparent glass plate2182is fixed to the microscope container2170in a state pressing the O-ring2181. The glass plate2182and the O-ring2181cooperate with the microscope container2170to constitute isolation means or an isolator to isolate the microscope device from the moisture of the culture device.

A communication pipe2183is fixed to the opening on the side surface of the microscope container2170. The communication pipe2183is an extensible bellows, and connects the inside of the microscope container2170to the outside of the incubator2164. The communication pipe2183is schematically drawn so that it extends in a direction vertical to the moving direction, that is, longitudinal direction of the microscope container2170, that is, in the lateral direction for convenience of drawing inFIG. 17, but it is actually provided so that it extends in parallel with the moving direction, that is, longitudinal direction of the microscope container2170. In addition, one communication pipe2183is drawn inFIG. 17, but two communication pipes2183are actually provided for air intake and exhaustion. The slide device2166comprises a fixed portion2184, a moving portion2185and a rolling portion2186, and the fixed portion2184is fixed to the incubator2164, and the moving portion2185is fixed to the microscope container2170.

The incubator2164is provided with a fan2231for the purpose of eliminating uneven temperature and humidity inside of the incubator2164. However, vibration due to the fan2231deteriorates an obtained image. Therefore, the controller2232performs blurring prevention control to stop the fan when an image is taken. The blurring prevention control may be started simultaneously with the lighting of the illumination device2167. This blurring prevention control makes it possible to obtain high-quality image without blurring the image due to the fan vibration.

Owing to the sealing structure using the O-ring2181, the infiltration of moisture into the microscope container2170is reduced in an environment with the highly humid incubator2164, and even a slight amount of infiltrated moisture is discharged outside by the communication pipes2183without causing rust and condensation in the microscope part. Moreover, the slide device2166is provided to make it easy to take the microscope container2170in and out of the incubator2164for easier cleaning, thereby enabling contamination prevention even in the cell culture where contamination of bacteria and molds is not preferred. Further, a slight amount of moisture infiltration is caused in a sealed portion with the O-ring, so that two communication pipes may be provided to introduce the outside air through one communication pipe and to compulsorily remove the moisture inside through the other communication pipe. This enables a more complete moisture measurement.

The culture microscope apparatus has a region I between the incubator2164and the microscope container2170, a region II within the microscope container2170, and a region III out of the incubator2164. In the region I, the temperature and humidity are maintained at a level equal to that in an environment inside a body of a human being, an animal or a plant. In the region II, the temperature is on a level equal that of the region I and the humidity is maintained at a normal humidity level. In the region III, the temperature and humidity are on a normal temperature and normal humidity level.

The regions I, II and III are located along the optical axis of the image-forming optical system of the observation device2168. The specimen2123is located in the region I.

The regions I and II are optically connected through the optically transparent glass plate2182, which is positioned between the regions I and II, so that light for the observation device2168, light from the specimen2123, passes through a boundary between the regions I and II.

The regions I and II, also in the present embodiment, satisfy the following conditional equations:
36° C.≦t1≦38° C., 90%≦h1≦100%
36° C.≦t2≦38° C., 50%≦h2≦80%
where t1indicates the temperature of the region I, h1indicates the humidity of the region I, t2indicates the temperature of the region II, and h2indicates the humidity of the region II.

The culture microscope apparatus of the present embodiment has the region II, in which the observation device2168is located, so that the adverse effect (especially condensation) on the observation device2168is effectively reduced.

In the present embodiment, the microscope container2170is taken in and out of the incubator2164by the slide device2166, but the mechanism to take the microscope container2170in and out is not limited thereto, and any known moving mechanism is applicable. Moreover, the moving mechanism such as the slide device2166may be omitted, and the microscope container2170may be manually taken in and out of the incubator2164.

In the present embodiment, the illumination device2167for the fluorescent observation is again the oblique illumination device, but may be the incident-light illumination device. That is, the illumination device2167for the fluorescent observation includes the objective lens2174and may have the configuration similar to those of the illumination devices described in the first embodiment to fourth embodiment.

While the embodiments of the present invention have been described above referring to the drawings, the present invention is not limited these embodiments, and various modifications and alterations may be made without departing from the sprit thereof.

The microscope device in the culture microscope apparatus is an inverted microscope in the above embodiments, but is not limited to it, that is, the microscope device may be an up-right microscope.