JP2005331889A - Fluorescence microscope and fluorescence observation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescence microscope or the like which gives a warning to a user so as to avoid discoloration or damage caused by the radiation of exciting light. <P>SOLUTION: The fluorescence microscope, where fluorescence dye is introduced into a sample being an observation object, the sample is arranged in an optical system and irradiated with the exciting light, and fluorescence excited by the exciting light and emitted is received so that a fluorescent image is formed, includes an exciting light source for emitting the exciting light to be radiated to the sample, a timer part for measuring the radiation time of the exciting light radiated by the exciting light source; a decision part for deciding whether or not the measured time measured by the timer part exceeds a predetermined reference value; and a warning part controlled by the decision part and performing warning operation for reducing the exciting light radiated to the sample from the exciting light source. Thus, the operator of the fluorescence microscope can discriminate whether or not the exciting light exceeding the reference value is radiated according as the warning is given or not, and adverse influence exerted on the sample by the excess of the exciting light is avoided. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a fluorescence microscope and a fluorescence observation method having a function of capturing and displaying a fluorescence image of a sample.

  Conventionally, fluorescence microscopes and laser microscopes have been used to observe the fine structure of cells, the localization of molecules, and the like. In a fluorescence microscope, a fluorescent molecule that specifically binds to a specific molecule of interest in a sample (referred to as a fluorescent probe or the like, for example, an antibody of a protein of interest covalently bound to a fluorescent molecule) In addition, we observe the distribution and movement of this molecule. An example of the epifluorescence microscope will be described with reference to FIG. The epi-illumination is an illumination method in which the light source light is irradiated through the objective lens 950 and the fluorescence from the sample W is observed through the objective lens 950. The illumination light (excitation light) and the observation light (fluorescence) use the same optical path of the objective lens 950, and the objective lens 950 also serves as a capacitor. In FIG. 9, the epifluorescence microscope uses a dichroic mirror 914 in order to achieve a configuration that realizes observation by epi-illumination. The dichroic mirror 914 is set together with an excitation filter 912 and an absorption (barrier) filter 916 in a box-like body generally called a dichroic cube (filter set). The light emitted from the light source is first cut off by the excitation filter 912, and only light having a wavelength that is absorbed by the fluorescent molecules of the fluorescent dye used is allowed to pass. This attenuates the background light that is an obstacle to observation. This excitation light is reflected at a right angle by a dichroic mirror 914 inclined at about 45 ° with respect to the optical axis, and reaches the sample W through the objective lens 950. The fluorescence emitted from the sample W travels in the opposite direction to the excitation light and reaches the dichroic mirror 914 through the objective lens 950. Since the dichroic mirror 914 has a property of reflecting light of a specific wavelength or less and passing light of a longer wavelength, fluorescence passes through this mirror. After passing through the fluorescence, the absorption filter 916 cuts the wavelength other than the fluorescence of interest and makes the background as dark as possible, and then guides the elapsed light to the eyepiece 9 (see Patent Document 1). Here, FIG. 10 shows a list of combinations of typical fluorescent dyes, their excitation filters, and absorption filters as combinations of fluorescent dyes and filters introduced into the sample. In FIG. 10, the reagent name (common name) of the fluorescent dye, the wavelength of the excitation light, and the wavelength of the absorption light are indicated by main peak values in the band.

In such fluorescence observation, since the fluorescence image is extremely weak light, it is necessary to irradiate the sample with very strong excitation light in order to obtain necessary fluorescence. However, in this case, in the case of a sample that easily fades, there is a disadvantage that the fluorescent fading is accelerated by excitation light, and in the case of a living sample, damage to the sample is also increased. Generally, as the excitation light is irradiated, the fluorescence intensity gradually decreases (dark blue), the contrast with the non-fluorescent part (background) decreases, and the fluorescent image cannot be captured correctly. End up. In particular, when observing living cultured cells for a long period of time called time-lapse photography, if the excitation light is irradiated too much, the cells themselves may be damaged and faded or killed. Therefore, in order to protect the sample from discoloration and damage, sufficient consideration must be given to the excitation light irradiation. Conventionally, however, the user can determine how much excitation light can be irradiated. It was difficult.
JP 2000-227556 A

  The present invention has been made to solve such conventional problems. A main object of the present invention is to provide a fluorescence microscope and a fluorescence observation method capable of avoiding discoloration and damage due to irradiation of excitation light.

  In order to achieve the above object, the fluorescence microscope of the present invention introduces a fluorescent dye into a sample to be observed, arranges it in an optical system, emits excitation light, and is excited and emitted by the excitation light. A fluorescence microscope that receives fluorescence and forms a fluorescence image, an excitation light source that emits excitation light to irradiate a sample, a timing unit that measures the irradiation time of the excitation light emitted by the excitation light source, and a timing unit A determination unit for determining whether or not the measured time exceeds a predetermined reference value, and a warning that is controlled by the determination unit to perform a warning operation to reduce excitation light irradiated on the sample from the excitation light source A part. Accordingly, the operator of the fluorescence microscope can determine whether or not the excitation light exceeding the reference value has been irradiated depending on the presence or absence of the warning, and can avoid the adverse effect on the sample due to excessive excitation light.

  In another fluorescent microscope of the present invention, the warning unit is disposed in the optical path of the excitation light irradiated on the sample from the excitation light source, and includes a blocking unit capable of blocking the excitation light irradiated on the sample. On the basis of the determination result, the excitation light is forcibly blocked by the blocking unit. Thus, when the excitation light exceeds the reference value, the excitation light is automatically blocked by the blocking unit, so that the sample is reliably protected from excessive excitation light.

  Furthermore, another fluorescent microscope of the present invention includes a warning display unit for warning that the level of the excitation light has reached the reference value or more, and based on the determination result of the determination unit, the warning unit Is configured to issue a warning by voice or / and character display and prompt a treatment to reduce the excitation light. As a result, when the excitation light exceeds the reference value, the user is notified of this, so that necessary measures can be taken and the sample can be protected.

  Furthermore, in another fluorescent microscope of the present invention, the timer unit measures the continuous irradiation time of the excitation light. Thus, the continuous irradiation time of the excitation light is automatically calculated, and a warning is obtained when the reference value is exceeded, so that the sample can be reliably protected.

  Furthermore, in another fluorescent microscope of the present invention, the timer unit calculates the cumulative irradiation time of the excitation light. Thus, since the influence on the sample can be estimated by the accumulated time of the excitation light, there is no need to record and add the troublesome observation time every time the fluorescence observation is performed, and an easy-to-use fluorescence microscope is realized.

  Furthermore, another fluorescent microscope of the present invention further includes a sample specifying unit that specifies a sample to be observed, and the time counting unit calculates the cumulative irradiation time for each sample specified by the sample specifying unit. As a result, the cumulative irradiation time can be calculated for each sample, so that even when observing multiple samples, it is possible to grasp the influence of excitation light for each sample and to ensure reliable fading prevention and sample protection. Become.

  Furthermore, another fluorescent microscope of the present invention further includes an observation condition designating unit for inputting the observation conditions of the sample, and the determination unit sets a predetermined reference value according to the observation conditions input from the observation condition designating unit. Configured to set. This makes it possible to calculate an appropriate excitation light tolerance reference according to the observation conditions, and to automatically set a reference value according to the sample to provide a user-friendly fluorescence microscope.

  Furthermore, in another fluorescent microscope of the present invention, the observation condition for setting a predetermined reference value is the type and / or pH concentration of the fluorescent dye introduced into the sample. As a result, it is possible to calculate an appropriate excitation light tolerance reference according to the type and pH concentration of the fluorescent dye as the observation condition parameter, and to automatically set the reference value according to the observation condition to provide a user-friendly fluorescence microscope. .

  Furthermore, another fluorescent microscope of the present invention introduces a fluorescent dye into a sample to be observed, arranges it in an optical system, irradiates it with excitation light, and receives fluorescence emitted by being excited by the excitation light. A fluorescence microscope that forms a fluorescent image, an excitation light source that emits excitation light that irradiates a sample, a timepiece that measures the irradiation time of the excitation light emitted by the excitation light source, and a time measured by the timepiece A display unit for displaying. As a result, the user can check the irradiation time of the excitation light on the display unit, so that necessary measures such as stopping or reducing the excitation light irradiation can be taken as the irradiation time elapses.

  Further, the fluorescence observation method of the present invention introduces a fluorescent dye into a sample to be observed, arranges it in an optical system, irradiates excitation light, receives fluorescence emitted by the excitation light, and receives fluorescence. A fluorescence observation method using a fluorescence microscope for forming an image, the step of specifying a sample observation condition, a step of setting a predetermined reference value based on the specified observation condition, and an excitation light source A step of counting the irradiation time from the time when the irradiation of the excitation light is started, and a step of determining whether or not the timed irradiation time exceeds a predetermined reference value and performing a warning operation when it is determined that the irradiation time has been exceeded. . Thereby, the operator of the fluorescence microscope can determine whether or not the excitation light exceeding the reference value has been irradiated depending on the presence or absence of the warning, and can avoid the adverse effect on the sample due to excessive excitation light.

  According to the fluorescence microscope and the fluorescence observation method of the present invention, it is possible to effectively prevent the occurrence of discoloration of the sample or damage to the sample itself as a result of irradiation with strong excitation light for a long time. This is because the fluorescence microscope and the fluorescence observation method of the present invention can notify the user of the irradiation time that affects the sample and warn the user by measuring the irradiation time of the excitation light. Therefore, the user can grasp the state of the sample from the excitation light irradiation time and can take appropriate measures to protect the sample appropriately.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. However, the embodiment described below exemplifies a fluorescence microscope and a fluorescence observation method for embodying the technical idea of the present invention, and the present invention specifies the fluorescence microscope and the fluorescence observation method as follows. do not do. In particular, the present specification by no means specifies the members shown in the claims as the members of the embodiments. Note that the size, positional relationship, and the like of the members shown in each drawing may be exaggerated for clarity of explanation. Furthermore, in the following description, the same name and symbol indicate the same or the same members, and detailed description thereof will be omitted as appropriate. Furthermore, each element constituting the present invention may be configured such that a plurality of elements are constituted by the same member and the plurality of elements are shared by one member, and conversely, the function of one member is constituted by a plurality of members. It can also be realized by sharing.

  FIG. 1 shows a block diagram of a fluorescence microscope according to an embodiment of the present invention. In the following example, using a fluorescence microscope, multicolor fluorescence observation is performed by introducing a plurality of fluorescent dyes (fluorescent dyes) into a sample W (also referred to as a specimen, a specimen, a sample, etc.) and staining and expressing in multiple colors. An example will be described. The fluorescence microscope 100 shown in this figure includes an excitation light source 48, a collector lens 54, a filter set 1, an objective lens 50, an imaging lens 52, an imaging unit 22, and a blocking unit 72. These members are arranged on a certain optical path. The excitation light source 48 emits excitation light that excites the fluorescent dye. For example, a high-pressure mercury lamp or a high-pressure xenon lamp is used. They emit a wide range of wavelengths of light. As the excitation light source 48, a light-emitting diode with low power consumption, small size, and high efficiency can be used. Moreover, members, such as an excitation light source, are unitized and a fluorescence microscope system can also be comprised combining a unit. The illumination light from these excitation light sources 48 is converted into a substantially parallel light beam by the collector lens 54 and introduced into the filter set 1 as excitation light. The collector lens 54 may be a fluorescent epi-illumination lens in the case of epi-illumination. In the following example, the illumination of the sample is the epi-illumination type, but the present invention is also applicable to other illumination methods such as transmission type illumination and total reflection illumination type. In the example of FIG. 1, only the imaging unit 22 is illustrated and a visual eyepiece is not provided, but it is needless to say that a visual mechanism may be provided. In this case, the fluorescence that has passed through the imaging lens can be selectively switched, reflected, and branched using a mirror or the like.

The filter set 1 is a combination of a single-pass filter and a mirror that selectively transmits light having a wavelength suitable for observation of a specific fluorescent dye, and as shown in the figure, an excitation filter 12, an absorption filter 16, and a dichroic mirror 14 are used. Is provided. Furthermore, the filter set 1 is configured so that a plurality of types can be switched. Each filter set 1 includes a combination of an excitation filter 12, an absorption filter 16, and a dichroic mirror 14 corresponding to the fluorescent dye to be used, and by switching these, different monochrome images can be taken. The plurality of filter sets 1 are set in the filter holder 56 and switched by the filter switching unit 18. The excitation light selected by the excitation filter 12 of the filter set 1 is reflected by the dichroic mirror 14, passes through the objective lens 50, and is projected onto the sample W. The objective lens 50 also serves as a condenser lens. The objective lens 50 can be exchanged according to the observation purpose, and can be configured to be able to switch between a plurality of objective lenses 50 by a screw type or the like, or by a revolver or the like.
(Dark room space 46)

  The sample W is placed on the sample placement unit 28. In general, observation of a faint light sample such as a fluorescent sample is performed in a dark room because disturbance light needs to be excluded. In the fluorescence microscope 100 according to the present embodiment, the optical path of the sample placement unit 28 and the optical system 10 is disposed in a dark room space 46 that is shielded from disturbance light, and the dark room space 46 is brought into a dark room state, thereby providing a dark room. Fluorescence observation can be done without preparing a, improving usability. As the sample placement unit 28, an XY stage or the like can be used, and it can be moved in the X-axis and Y-axis directions. Further, the sample mounting portion 28 can be moved in the vertical direction (Z-axis direction), so that the focus can be adjusted by changing the relative distance from the optical system 10.

In FIG. 1, among the fluorescent dyes included in the sample W, the fluorescent dye corresponding to the irradiated excitation light emits fluorescence, and this fluorescence passes through the objective lens 50 and enters the filter set 1, and passes through the dichroic mirror 14. pass. Thus, the dichroic mirror 14 reflects the illumination light and transmits the fluorescence. Further, the fluorescent light passes through the absorption filter 16 and selectively absorbs light components other than fluorescent light such as illumination light. The absorption filter 16 is also called a barrier filter, and is disposed closer to the fluorescent image forming surface than the dichroic mirror 14. The light exiting the filter set 1 passes through the imaging lens 52 and enters the imaging unit 22. The imaging unit 22 is disposed at a position conjugate with the focal plane of the objective lens 50. The imaging unit 22 converts the fluorescence into an electrical signal, an image is generated based on the signal, and is displayed on the display unit 24. For this reason, the imaging unit 22 includes an imaging element, and a semiconductor imaging element such as a CCD camera can be preferably used. The CCD cameras are arranged two-dimensionally and can simultaneously image one screen without sequentially scanning the screen as in a laser microscope. Since the CCD camera can improve noise characteristics by cooling, a CCD camera equipped with a mechanism cooled by a Peltier element, liquid nitrogen, or the like may be used. As described above, the fluorescence microscope can automatically switch the single-pass filter set 1 by the filter switching unit 18 and can simultaneously display the monochromatic image captured by each filter set 1 and the superimposed image obtained by superimposing these images.
(Blocking part 72)

Further, the fluorescence microscope includes a control unit 26 and a blocking unit 72 connected to the imaging unit 22. The control unit 26 is connected to a computer and a display unit that are externally connected devices. A blocking unit 72 is provided between the excitation light source and the collector lens. The blocking unit 72 is disposed on the optical path of the excitation light emitted from the excitation light source, and forcibly blocks the excitation light when inserted into the emission light path. The blocking unit 72 can be switched between an open position and a blocking position so as to selectively pass and block the excitation light by a shutter type, open / close type, or the like. The blocking unit 72 is controlled by the control unit 26, and the control unit 26 controls the operation of the motor that switches between open / close. In general, when the excitation condition or the observation condition is changed, the blocking unit 72 blocks the excitation light, and is opened so as to irradiate the excitation light again after switching the excitation filter, the dichroic mirror, and the absorption filter. In the example of FIG. 1, the blocking unit 72 is disposed between the excitation light source and the collector lens. However, the blocking unit 72 is not limited to this example. For example, a blocking unit may be disposed between the collector lens and the filter set.
(Control unit 26)

The control unit 26 measures the irradiation time of the excitation light emitted from the excitation light source, determines whether or not a predetermined reference value is exceeded, and performs a warning operation according to the determination result. In other words, the time during which the excitation light is irradiated to the sample is measured, and when the excitation is continued for a certain period (reference value) continuously, or the same sample is irradiated with the excitation light. A warning action is performed when the accumulated time exceeds the reference value. For this reason, the control part 26 is provided with the timer which time-measures as a time measuring part. When the irradiation of the excitation light is disclosed, the timer starts timing, and the continuous irradiation time of the excitation light is counted. When the excitation light irradiation is stopped, the timing is stopped. Thereby, not only continuous irradiation time but the cumulative time of discrete irradiation can also be timed.
(Sample identification part)

Furthermore, the control unit 26 can measure the excitation light irradiation time for each sample. For example, when fluorescence observation is performed by switching or exchanging a plurality of samples, the state of each sample can be accurately grasped by storing the irradiation time of excitation light for each sample. The sample is specified by the sample specifying unit. For example, each time the user switches the sample, the method of manually instructing the switching of the sample, or the fluorescent microscope side automatically detects the switching of the sample and discriminates the switched sample based on the characteristics The irradiation time is held for each sample. As the feature quantity used for automatic discrimination of the sample, image recognition of the sample shape, fluorescence brightness, wavelength, and the like can be used. In addition, sample switching can be performed by, for example, detecting the opening / closing of a panel for inserting a sample into a fluorescence microscope, detecting the exchange of a preparation in which a sample placed on the sample placement unit is set, using a sensor, or the like. For example, a method of detecting the initial movement of the camera or manually instructing the user to switch the sample can be used. As described above, in the case of automatically discriminating the sample, when the fluorescence microscope detects the switching of the sample, the feature amount of the sample is detected to discriminate the sample. The sample specifying unit is realized by the control unit 26. The control unit 26 can be realized by a gate array such as a microprocessor (MPU), CPU, LSI, FPGA, ASIC or the like. In FIG. 1, the control unit 26 corresponds to a time measuring unit, a determination unit, and a sample specifying unit. It is also possible to cause a computer, which is an externally connected device connected externally to the fluorescence microscope, to execute functions such as a time measuring unit, a determination unit, and a sample specifying unit.
(Standard value)

Next, a reference value of the irradiation time of excitation light corresponding to the sample is determined. The reference value depends on observation conditions such as the intensity and pH concentration of the fluorescent dye introduced into the sample. Therefore, in the present embodiment, an observation condition is designated in advance from the observation condition designation unit, and a reference value is set according to the designated observation condition. The reference value is determined by measuring the continuous irradiation time or / and the cumulative irradiation time of the excitation light and reaching the reference value. However, the continuous irradiation time and the cumulative irradiation time of the excitation light can be individually specified. Both the continuous irradiation time and the cumulative irradiation time are individually measured by the time measuring unit, and a warning operation is performed when either one exceeds a corresponding reference value. In addition, it can also be set as the structure which measures only any one of continuous irradiation time or accumulated irradiation time in a time measuring part, and determines the reference value corresponding to the measured irradiation time. In this case, the user designates in advance whether to monitor the continuous irradiation time or the cumulative irradiation time. Moreover, it can also be set as the fluorescence microscope which can time only any one.
(Observation condition designation part)

  In the observation condition designating unit, the user designates parameters of the fluorescence observation conditions. In this example, the type and / or pH concentration of the fluorescent dye introduced into the sample. Based on this designation, the control unit 26 determines an appropriate reference value for the excitation light irradiation time. In the determination method, a lookup table in which a reference value corresponding to a combination of observation condition parameters is defined in advance is stored in the control unit 26, and the reference value is determined based on the specified observation condition based on the table. Thereby, it is possible to automatically set a reference value according to the observation condition and to make the fluorescence microscope easy to use. In the example of FIG. 1, an input device of a computer that is an externally connected device corresponds to an observation condition specifying unit.

The observation condition designating unit may be configured to automatically detect the observation condition on the fluorescence microscope side, in addition to designating by the user. For example, the fluorescence microscope detects the intensity and pH concentration of the fluorescent dye used, discriminates the fluorescence observation conditions accordingly, and further calculates a reference value based on the fluorescence observation conditions. In this configuration, since the fluorescence microscope automatically performs calculations from necessary condition settings, the user can be made a fluorescence microscope with an excitation light irradiation time warning function that is easy for beginners to handle without being conscious of these. Further, automatic detection by a fluorescence microscope and manual designation by a user can be appropriately combined. For example, the fluorescence microscope automatically calculates the wavelength from the detected fluorescent color, and based on this, the fluorescent dye is inferred and displayed as a list on the display unit. The user selects an appropriate fluorescent dye from this list. As a result, erroneous detection can be avoided, and more reliable and accurate observation conditions can be specified, a sample can be specified, and a reference value can be selected.
(Warning action)

When the set reference value exceeds the continuous irradiation time or the accumulated time of the irradiation time measured by the control unit 26, the control unit 26 instructs to perform a warning operation. Here, as a warning action, a message indicating that the reference value has been exceeded is displayed on the display unit, voice guidance, warning sound, flashing light, action of a warning indicator provided as an individual member, or a combination thereof Is mentioned. As an example of the warning operation, an example of a warning message displayed on the display unit is shown in FIG. For example, when the continuous irradiation time exceeds the reference value, as shown in FIG. 2 (a), a warning message 74 is displayed such as “Excitation light continuous irradiation time exceeds XX minutes”, and the cumulative irradiation time. Similarly, when the value exceeds the reference value, as shown in FIG. 2B, the warning message 74B is displayed as “the cumulative irradiation time of excitation light has exceeded OO minutes” or the like. As a result, the user can recognize that the irradiation of the excitation light is approaching the limit, and thus can take necessary measures such as early termination or cancellation of the fluorescence observation and stop of the irradiation of the excitation light. It is also possible to forcibly stop the irradiation of excitation light by closing the blocking portion 72. This ensures that the sample can be protected. In this way, the state of the sample can be ascertained by measuring the excitation light irradiation time, so that the user can use the minimum necessary focus, field-of-view adjustment, and imaging to suppress the fading of the sample as much as possible. The operation time is as short as possible.
(Display elapsed time)

  Further, in addition to a configuration in which a warning is given when a predetermined reference value is reached, the continuous time and cumulative time of excitation light irradiation can be displayed on the display unit. A display example of the display unit according to this embodiment is shown in FIG. In FIG. 3A, the irradiation time measured by the time measuring unit is displayed on the display unit 24 as the irradiation time display unit 76, and the reference time is also written as “warning time”. Thereby, the user can grasp the current state of the excitation light. Further, the remaining time until the reference time is reached may be displayed. Further, in the example of FIG. 3B, the irradiation time is displayed as a graph display instead of a numerical value as the irradiation time display section 76B. In this example, the irradiation time display unit 76B is used as a level meter to display the irradiation time, and a mark or line such as a triangle is displayed at a position corresponding to the reference value, and the region is changed to red in an area exceeding the reference value. Thus, the relationship with the reference value is expressed visually. In this way, by displaying the elapsed time in the state of the level meter or the indicator, it is possible to visually recognize the damage state to the sample. In addition to updating these displays in real time, they can also be updated at regular time intervals. Furthermore, it goes without saying that the above numerical display and graph display can be combined. Furthermore, a display of elapsed time and a warning operation may be combined. For example, the display as shown in FIGS. 3A and 3B may be performed when the irradiation time reaches a reference value. Thus, by displaying the elapsed time constantly, the user can always grasp the state of the sample. In general, a fluorescent dye introduced into a sample is dark and easily fades with time, so that a quick operation is required. For this reason, by notifying the user that the irradiation time has passed or that the specified reference value has been exceeded, the user can grasp the sample state relative to the danger level and consider discoloration and damage to the sample. In addition, fluorescence observation can be performed.

Hereinafter, a fluorescence observation method that performs a warning operation using the fluorescence microscope will be described based on the flowchart shown in FIG. First, after initialization, the control unit 26 calculates a reference value in step S1. Specifically, an observation condition is designated by the observation condition designation unit, and the control unit 26 selects a reference value based on the designated observation condition. Furthermore, when recording irradiation time for every sample with respect to a some sample, a sample specific | specification part specifies a sample. Next, when the irradiation of excitation light is started under the designated observation conditions, the timer of the timer unit starts measuring time in step S2. In step S3, the control unit 26 determines whether or not the measured time has reached the reference value. If the reference value has not been reached, step S3 is looped. When the reference value is reached, the process proceeds to step S4, and a predetermined warning operation such as display of a warning message or blocking of excitation light is performed.
(Filter set 1)

  The filter set 1 includes a set of an excitation filter 12, an absorption filter 16, and a dichroic mirror 14 in a box-like body generally called a dichroic cube. In the filter set 1, the combination of the excitation filter 12, the dichroic mirror 14, and the absorption filter 16 is determined according to the fluorescent dye introduced into the sample W. A combination of single-pass bandpass filters is determined so as to extract light of a desired wavelength component and exclude other wavelength components so that only the color expressed by the fluorescent dye can be correctly observed. Therefore, the filter set 1 to be used is determined according to the fluorescent dye to be used. In general, filter sets 1 having different fluorescent colors are used, and combinations of colors according to fluorescent dyes such as RGB and CMY can be appropriately employed. Here, the combination of the filter set and the fluorescent dye is determined by the fluorescent dye, its excitation light, and the fluorescence wavelength, and is appropriately selected according to the fluorescence observation based on the list shown in FIG. The plurality of filter sets 1 are configured to be switchable by a filter switching unit 18. The plurality of filter sets 1 are set in the filter holder 56, and the filter switching unit 18 causes any one of the plurality of filter sets 1 to be set on the optical path. The filter switching unit 18 may be a turret that switches the filter set 1 in a rotating manner or a sliding manner by driving a motor or the like. Control of the filter switching unit 18 is set by the switching setting unit 20. A necessary set of excitation filter 12, absorption filter 16, and dichroic mirror 14 can be switched at once using the filter set 1, and the switching operation portion can be integrated into one place to facilitate high-speed operation and maintenance. it can. However, without using a filter set consisting of an excitation filter, an absorption filter, and a dichroic mirror, individual switching means for individually switching multiple excitation filters, absorption filters, and dichroic mirrors are provided, and each switching means is linked. Thus, a predetermined set of excitation filters, absorption filters, and dichroic mirrors may be arranged on the optical path. Furthermore, it is possible to switch the filters and take an image by changing the optical path with a mirror or the like without moving the filters.

In addition, as a method of performing multicolor fluorescence observation using a fluorescence microscope, a method of using a dual or triple bandpass filter that can simultaneously observe a plurality of fluorescent dyes by mounting an imaging device such as a CCD camera on the fluorescence microscope, A method of switching and using a single-pass (single color) filter set corresponding to each fluorescent dye to be used can be used as appropriate.
(Display unit 24)

  The display unit 24 is a display that displays an image captured by the optical system 10. The display constituting the display unit 24 is a monitor capable of high-resolution display, and a CRT, a liquid crystal panel, or the like is used. The display unit 24 may be displayed on an external connection device 58 connected to the fluorescence microscope 200 as shown in FIG. 5 in addition to a monitor incorporated in or attached to the fluorescence microscope. For example, when the computer 58A is used as the external connection device 58, the function of the display unit can be realized by the monitor 24A of the computer 58A. Of course, a plurality of display units can be used on both the fluorescence microscope 200 side and the external connection device 58 side.

  FIG. 6 shows an example of a user interface screen of a fluorescence microscope image display program for setting a display form of an image captured by the fluorescence microscope. As shown in FIG. 5, this program is installed in a computer 58A connected to the fluorescence microscope 200, receives image data picked up by the fluorescence microscope 200, displays it on the monitor 24A functioning as a display unit, and according to the settings. The operation of the fluorescence microscope 200 is controlled so as to obtain a desired image display. FIG. 5 shows an example of the configuration. In the present invention, various configurations can be used for the fluorescence microscope system. For example, the control unit 26, the operation unit, the monitor, etc. are provided in the fluorescence microscope itself without connecting external devices. Stand-alone settings and operations can be completed. Alternatively, a plurality of fluorescent microscopes can be connected with one computer, and can be operated in conjunction or independently.

An example of the display screen of the display unit 24 is shown in FIG. 6 for the GUI image of the fluorescence microscope image display program. As shown in this figure, the display unit 24 includes a plurality of image display areas G. Different images can be displayed in each image display area G, and a plurality of images can be displayed and compared at the same time. In particular, in the present embodiment, one of the image display areas G can be used as a superimposed image display area O, and a superimposed image in which images observed by the respective filter sets are superimposed can be displayed. Thereby, the image observed with each filter set and these superimposed images can be compared on the same screen. In particular, as will be described later, each image can be displayed almost in real time in the present embodiment, so that the state of the sample under various conditions can be easily observed. In the example of FIG. 6, four image display areas G are used, and one of them is used as a superimposed image display area O. Of course, the image display area can be three or less, or five or more. Preferably, by setting the number of filter sets set in the filter holder to the number of image display areas added for displaying superimposed images, all filter sets and these superimposed images can be observed simultaneously on one screen. . Of course, any selected screen can be displayed in an enlarged manner, or the enlarged display screen can be displayed for each filter set or by switching to a superimposed image. Also, it is not necessary to display all images on the same screen, and each image can be displayed in a separate window or switched to full screen display. Thus, the display mode can be changed as appropriate according to the number of filter sets, the purpose of observation, the user's preference, and the like. In the example of FIG. 6, three filter sets respectively corresponding to the fluorescent dyes 1 to 3 are set in the filter holder 56, and each single color image picked up by switching these and a superimposed image in which the single color images are superimposed are displayed. An example is shown.
(Image adjustment unit 40)

  In addition, the setting screen illustrated in FIG. 6 includes an image adjustment unit 40 that adjusts a captured image. In the example of the setting screen shown in this figure, switches for adjusting image adjustment parameters such as position, height, magnification, brightness, and contrast for visual field movement as the image adjustment unit 40 are displayed in the image display region G. It is provided on the right side. These image adjustment parameters can be automatically set optimally in addition to being adjusted by the user. For example, a “one-touch auto” button for automatically adjusting the exposure time and a “one-touch focus” button for automatically adjusting the focus are provided. Among the adjustments of each image adjustment parameter, the position adjustment is a movement in the XY direction, and the viewpoint of the image being displayed can be moved. For example, the position, that is, the viewpoint can be moved by dragging an arbitrary position on the screen of the image display area G with a mouse and moving it in a desired direction. The sample placement unit 28 is moved in the XY directions according to the movement amount of the mouse. It can also be moved using the up / down / left / right buttons or the cross button. Further, a guide for displaying in which part of the displayable area the currently displayed viewpoint can be provided.

  The height is adjusted in the Z direction, that is, the relative distance between the sample W and the optical system 10 (objective lens 50), thereby adjusting the focus of the image. By adjusting the height with the height designation section, the sample placement section 28 is moved up and down. Adjustments such as height and magnification can be made continuously and visually using sliders, level meters, scales, and the like. In the example of FIG. 6, a slider 32A is used as the height designation portion. In either case, the sample placement unit 28 is moved for adjustment, but it goes without saying that the same result can be obtained even if the optical system 10 is moved.

The magnification is adjusted by the imaging lens 52. The imaging lens 52 is a lens that images the fluorescence from the absorption filter 16 of the filter set 1, and is disposed between the filter set 1 and the imaging unit 22. In this example, the image forming lens 52 is a zoom variable power lens, and the magnification can be continuously varied from 10 times to 100 times, for example, by using one image forming lens 52. Note that the magnification, numerical aperture, working distance, and the like of the objective lens and the imaging lens are set to appropriate values according to the observation purpose and conditions. The fluorescent image formed by the imaging lens 52 is projected onto the imaging unit 22, converted into an electrical signal, and displayed on the externally connected display unit 24. The image data of the fluorescent image is captured and processed by an external connection device 58 such as a computer 58A. Processing conditions and processing results are displayed on the display of the computer 58A. The computer display 58A can also be used as a display unit. The observer can observe the fluorescent image on the display unit, and further operates the computer 58A to perform a desired process on the fluorescent image, and can observe the result on the display unit or the display.
(Inverted fluorescence microscope)

In the example of FIG. 1, an example of an upright fluorescent microscope has been described, but it goes without saying that the present invention can be similarly applied to an inverted fluorescent microscope. FIG. 7 shows a block diagram of an inverted fluorescence microscope according to still another embodiment of the present invention. The inverted fluorescent microscope has a structure in which the objective lens 50, the filter set 1, the imaging lens 52, and the imaging unit 22 are sequentially arranged below the sample placement unit. As shown in FIG. 7, in the inverted fluorescent microscope, the configuration of the inverted fluorescent microscope can be simplified by omitting the visual eyepiece. In a conventional inverted fluorescence microscope, the fluorescence obtained through an objective lens or filter set provided below the sample placement unit is polarized upward in the optical path with a polarizing mirror, etc. In this way, the configuration is complicated because the optical path is bent and extended in this manner, which increases the size of the system and increases the cost. Therefore, by eliminating such an eyepiece and connecting a display unit that displays an image captured by the imaging unit to the outside, the system can be simplified and the apparatus can be downsized.
(Control system 64)

  Next, an example of a block diagram constituting the control system 64 of the fluorescence microscope is shown in FIG. In this figure, detailed display of the optical system 10 is omitted. As shown in this figure, the fluorescence microscope has, as an imaging system 66, a stage 28A that is one form of the sample placement unit 28 on which the sample W is placed, a stage elevator 30A that moves the stage 28A, and an excitation light source 48 ( An optical system 10 that excites the fluorescent dye with excitation light irradiated to the sample W placed on the stage 28A from (not shown in FIG. 8) and forms an image of the fluorescence on the imaging unit 22, and the imaging unit 22, a CCD 22 </ b> A that electrically reads fluorescence incident from the sample W fixed on the stage 28 </ b> A via the optical system 10 for each of the two-dimensionally arranged pixels, and a CCD control that drives and controls the CCD 22 </ b> A. Circuit 22B. The stage elevator 30A is a form of the height adjustment unit 30 and includes, for example, a stepping motor 30a and a motor control unit 30b that controls the elevation of the stepping motor 30a. The stepping motor 30a moves the stage 28A in the Z-axis direction in the optical axis direction and the XY direction that is in a plane perpendicular to the optical axis direction.

  In addition, as a control system 64 for controlling the imaging system 66, an interface unit 68 for exchanging electrical signals such as data with the imaging system 66 by means such as communication, and an image electrically read by the imaging unit 22 A memory unit 34 for holding data, a display unit 24 for displaying captured and synthesized images, various settings, and the like, and input, setting, and other operations based on a screen displayed on the display unit 24 The operation unit 70 for performing the operation and the imaging system 66 is controlled according to the conditions set in the operation unit 70 to perform imaging, and the obtained image data is combined to generate a stereoscopic image, or image processing and other various processes The control part 26 which performs is provided. The imaging system 66 and the control system 64 described above can be included in the fluorescence microscope to complete the operation only with the fluorescence microscope apparatus. As shown in FIG. 5, the external connection device 58 such as a computer 58A is connected to the fluorescence microscope 200. , The fluorescence microscope 200 can function the imaging system, and the external connection device 58 can function the control system. Further, the imaging system and the control system do not strictly distinguish constituent members. For example, a memory unit may be provided in the imaging system, or conversely, a CCD control circuit or a motor control unit may be provided in the control system.

  The operation unit 70 is connected to a fluorescent microscope or a computer constituting the fluorescent microscope system by a wired or wireless connection, or is fixed to the computer. Examples of the general operation unit include various pointing devices such as a mouse, keyboard, slide pad, track point, tablet, joystick, console, jog dial, digitizer, light pen, numeric keypad, touch pad, and accu point. In addition to the operation of the fluorescence microscope image display program, these operation units can be used for the operation of the fluorescence microscope and its peripheral devices. Furthermore, using a touch screen or touch panel on the display itself that displays the interface screen, the user can directly input and operate the screen by hand, or use voice input or other existing input means, Or these can also be used together. In the example of FIG. 8, the operation unit 70 includes a mouse (see FIG. 5). The mouse can be used to operate the slider 32A, which is the height designation unit 32, and to perform various operations such as focusing on the image. In this way, the display unit 24 displays the operation menu, settings, and the like together with the image, and by selecting the operation item and performing the operation on the image, the user can accurately grasp the operation content and state, and can prevent an operation error. In addition, an operational system that is sensory and easy to operate is realized.

  In the fluorescence microscope and fluorescence observation method of the present invention, for example, a patient's serum and cell nucleus are reacted, a fluorescent label is added, the wellbore is observed with a fluorescence microscope, and positive or negative is determined by the fluorescence of the wellbore. It can be used for fluorescent antibody testing.

It is a block diagram which shows the fluorescence microscope which concerns on one embodiment of this invention. It is an image figure which shows an example of the warning message displayed on a display part. It is an image figure which shows the other example of the warning message displayed on a display part. 5 is a flowchart of a fluorescence observation method according to an embodiment of the present invention. It is a block diagram which shows the fluorescence microscope system which concerns on other embodiment of this invention. It is an image figure which shows an example of the display screen of a display part. It is a block diagram which shows the fluorescence microscope which concerns on other embodiment of this invention. It is a block diagram which shows the control system of the fluorescence microscope which concerns on other embodiment of this invention. It is a block diagram which shows the conventional epifluorescence microscope. It is a list figure showing typical fluorescent pigment, its excitation light, and fluorescence wavelength.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100, 200 ... Fluorescence microscope 1 ... Filter set 9 ... Eyepiece 10 ... Optical system 12 ... Excitation filter 14 ... Dichroic mirror 16 ... Absorption filter 18 ... Filter switching part 20 ... Switching setting part 22 ... Imaging part; 22A ... CCD; ... CCD control circuit 24 ... display unit; 24A ... monitor 26 ... control unit 28 ... sample placement unit; 28A ... stage 30 ... height adjustment unit; 30A ... stage lifter 30a ... stepping motor; 30b ... motor control unit 32 ... Height designation section; 32A ... Slider 34 ... Memory section 40 ... Image adjustment section 42 ... Color correction section 44 ... Indicator; 44B ... Setting indicator 46 ... Dark room space 48 ... Excitation light source 50 ... Objective lens 52 ... Imaging lens 54 ... Collector Lens 56 ... Filter holder 58 ... External connection device; 58A ... Computer 64 Control system 66 ... Imaging system 68 ... Interface unit 70 ... Operation unit 72 ... Blocking unit 74, 74B ... Warning message 76, 76B ... Irradiation time display unit 912 ... Excitation filter; 914 ... Dichroic mirror; 916 ... Absorption filter W ... Sample; G: Image display area; O: Superposed image display area

Claims (11)

A fluorescence microscope that introduces a fluorescent dye into a sample to be observed, arranges it in an optical system, irradiates excitation light, receives fluorescence emitted by the excitation light, and forms a fluorescent image. ,
An excitation light source that emits excitation light to irradiate the sample;
A timing unit for measuring the irradiation time of the excitation light emitted by the excitation light source;
A determination unit for determining whether the time measured by the time measurement unit exceeds a predetermined reference value;
A warning unit that is controlled by the determination unit and performs a warning operation for reducing excitation light irradiated on the sample from the excitation light source;
A fluorescence microscope comprising:   2. The fluorescence microscope according to claim 1, wherein the warning unit includes a blocking unit that is arranged in an optical path of excitation light irradiated on the sample from the excitation light source and that can block excitation light irradiated on the sample. The fluorescence microscope is configured to forcibly block excitation light by the blocking unit based on a determination result of the determination unit.   The fluorescence microscope according to claim 1 or 2, wherein the warning unit includes a warning display unit for warning that the level of excitation light has reached a reference value or more, and the determination result of the determination unit The fluorescent display is characterized in that the warning display unit issues a warning by voice or / and character display and prompts a treatment for reducing the excitation light.   The fluorescence microscope according to claim 1 or 2, wherein the warning unit includes a warning display unit for warning that the level of excitation light has reached a reference value or more, and the determination result of the determination unit The fluorescent display is characterized in that the warning display unit issues a warning by voice or / and character display and prompts a treatment for reducing the excitation light.   4. The fluorescence microscope according to claim 1, wherein the timekeeping unit measures a continuous irradiation time of the excitation light. 5.   4. The fluorescence microscope according to claim 1, wherein the time measuring unit calculates a cumulative irradiation time of the excitation light. 5.   The fluorescence microscope according to claim 6, further comprising a sample specifying unit that specifies a sample to be observed, and the time counting unit calculates a cumulative irradiation time for each sample specified by the sample specifying unit. Fluorescence microscope characterized by performing. The fluorescence microscope according to any one of claims 1 to 7, further comprising an observation condition designating unit for inputting an observation condition of the sample,
The fluorescence microscope, wherein the determination unit is configured to set a predetermined reference value according to an observation condition input from the observation condition designating unit.   9. The fluorescence microscope according to claim 8, wherein the observation condition for setting the predetermined reference value is the type and / or pH concentration of the fluorescent dye introduced into the sample. A fluorescence microscope that introduces a fluorescent dye into a sample to be observed, arranges it in an optical system, irradiates excitation light, receives fluorescence emitted by the excitation light, and forms a fluorescent image. ,
An excitation light source that emits excitation light to irradiate the sample;
A timing unit for measuring the irradiation time of the excitation light emitted by the excitation light source;
A display unit for displaying the time measured by the time measuring unit;
A fluorescence microscope comprising: A fluorescent dye was introduced into a sample to be observed, placed in an optical system, irradiated with excitation light, and a fluorescence microscope was used to receive fluorescence emitted by the excitation light and form a fluorescence image. A fluorescence observation method,
Specifying the observation conditions of the sample;
Setting a predetermined reference value based on designated observation conditions;
A step of measuring the irradiation time from the point of time when excitation light irradiation is started on the sample with the excitation light source;
Determining whether the measured irradiation time exceeds a predetermined reference value, and performing a warning operation when it is determined that it has exceeded;
A fluorescence observation method comprising: