JP3999324B2 - Fluorescence microscope device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a fluorescence microscope apparatus that is used when observing the state of a specimen such as a biological tissue in the fields of medicine and biology, and in particular, photographs fluorescence of a plurality of wavelength regions emitted from the specimen.
[0002]
[Prior art]
In general, this type of fluorescence microscope apparatus is used to detect fluorescently labeled proteins, genes, and the like on tissue cells of organisms in medicine, biology, and other fields.
[0003]
In particular, in recent years, when a specimen is fluorescently labeled, even a substance that emits only weak fluorescence has been frequently used to investigate the positional relationship with other substances by multiple staining with a plurality of fluorescent labels. .
[0004]
Recently, a fluorescent dichroic mirror for multiple staining has been used in a fluorescence microscope apparatus for observing such multiple stained specimens in order to identify the fluorescence wavelength of each staining.
[0005]
However, when preparing multiple-stained specimens, it is difficult to balance the brightness of each fluorescence with each fluorescence label obtained with a fluorescence microscope because the brightness of these fluorescence cannot be confirmed. Yes.
[0006]
For example, in JP-A-8-320437, a filter having a variable transmission characteristic with respect to a wavelength is inserted on the illumination optical path of a specimen, the amount of excitation light with respect to the fluorescent label is adjusted, and the fluorescence of each fluorescent label is adjusted. The brightness is balanced.
[0007]
On the other hand, in fluorescence photography using a fluorescence microscope apparatus, since the illuminance of fluorescence is low, the film is used in the illuminance region where the low illuminance irregularity characteristic is exhibited, resulting in excessive or insufficient film sensitivity density, due to proper exposure I can't shoot.
[0008]
In particular, during fluorescence observation, the fluorescent specimen exhibits a characteristic color that has a peak at its emission wavelength. Therefore, when using a color film, three types of emulsion layers R (red), G (green), and B (blue) are used. However, there is a problem that a photo with proper exposure cannot be obtained depending on the color of the fluorescent dye used.
[0009]
For this reason, for example, in JP-A-5-165079, the fluorescence wavelength of a specimen is detected, exposure correction is performed by changing the exposure time of the camera, and good photography is performed.
[0010]
[Problems to be solved by the invention]
However, in the method of balancing the brightness of each fluorescence as in the technique of the above-mentioned JP-A-8-320437, the brightness balance of each fluorescence is manually adjusted based on the observation light from the specimen. Skill is required for the adjustment work, and the adjustment takes time because it is a manual work.
[0011]
In general, fluorescent dyes are dark and easily faded over time, and therefore require rapid work.
In the technique disclosed in Japanese Patent Application Laid-Open No. 5-165079, the brightness of the fluorescence obtained from the specimen is detected for each wavelength and the exposure time is corrected. Since the exposure is repeated every time, multiple exposures must be performed, taking a long time for taking a picture, and causing a problem of fading of the fluorescent dye.
[0012]
Therefore, the present invention provides a fluorescence microscope apparatus that can simplify the work and reduce the fading of the specimen to observe the multiple stained specimen, and can balance the brightness of fluorescence by each fluorescent label. For the purpose.
[0013]
[Means for Solving the Problems]
According to claim 1, in the fluorescence microscope apparatus for irradiating the multiple-stained specimen with excitation light and observing the fluorescence emitted from the specimen, the detection element for detecting the brightness of the fluorescence emitted from the specimen, and the specimen Dimming means for dimming the fluorescence emitted from each wavelength, and dimming so that the brightness of the fluorescence observed based on the fluorescence for each wavelength detected by the detection element is equal for each wavelength A fluorescence microscope apparatus comprising control means for controlling the means.
[0014]
According to a second aspect of the present invention, in the fluorescence microscope apparatus according to the first aspect, the dimming means is a filter whose spectral transmission characteristics vary depending on the incident angle of light.
According to claim 3, in the fluorescence microscope apparatus according to claim 1, the dimming means is a filter having different transmittance in a specific wavelength region.
[0015]
DETAILED DESCRIPTION OF THE INVENTION
(1) Hereinafter, a first embodiment of the present invention will be described with reference to the drawings.
FIG. 1 is a configuration diagram of a fluorescence microscope apparatus.
A dichroic mirror 2 is arranged on the optical path of light emitted from the illumination light source 1. The dichroic mirror 2 has an action of reflecting only light in a specific wavelength region necessary for exciting the fluorescent dye out of the light emitted from the light source 1.
[0016]
An objective lens 3 is disposed on the reflected light path of the dichroic mirror 2. For example, a specimen 4 that is double-stained with each fluorescent label is disposed at the focusing position of the objective lens 3.
[0017]
In addition, a camera 6 is disposed on the light transmission path from the specimen 4 of the dichroic mirror 2 via a quick return mirror 5. The quick return mirror 5 is inserted into the transmission optical path of the dichroic mirror 2 to bend the optical path, or separated from the transmission optical path of the dichroic mirror 2 to transmit the specimen image from the specimen 4 to the camera 6 side. ing.
[0018]
A light control means 7 is disposed between the light source 1 and the dichroic mirror 2.
The dimming means 7 has a function of dimming the fluorescence emitted from the specimen 4 for each wavelength. For example, a filter 7a whose spectral transmission characteristics change depending on the incident angle of light is used.
[0019]
FIG. 2 is a diagram showing the characteristics of such a filter. The filter 7a is provided so as to be rotatable so that the incident angle is set to 0 ° or 45 ° with respect to the traveling direction of the light from the light source 1. If it is on the long wavelength side and the incident angle of the light beam is 45 °, the transmission band for the light beam moves to the short wavelength side.
[0020]
On the other hand, on the reflected light path of the quick return mirror 5, a two-dimensional area sensor 9 is disposed at a position conjugate with the sample surface via the imaging lens 8. The two-dimensional area sensor 9 has a function as a detection element that outputs a luminance signal corresponding to the brightness of the sample image projected by the imaging lens 8.
[0021]
The control device 10 sets incident angles to the filter 7a of the light control means 7 to 0 ° and 45 °, and inputs each luminance signal output from the two-dimensional area sensor 9 at these incident angles. It has a function of controlling the angle of the filter 7a of the light control means 7 so that the brightness of the fluorescence observed based on the brightness signal for each wavelength in the fluorescence from the sample 4 is equal for each wavelength from the luminance signal. Yes.
[0022]
Specifically, the control device 10 includes functions of a memory unit 11, a comparison unit 12, and an output unit 13. Among these, the memory unit 11 stores the luminance signal output from the two-dimensional area sensor 9. The comparison unit 12 is connected to external information input means 14.
[0023]
The external information input means 14 stores correction values for correcting the low illuminance failure characteristics in R, G, and B depending on the type of film used for the camera 6.
The comparison unit 12 inputs each luminance signal output from the two-dimensional area sensor 9 when the incident angle to the filter 7a of the light control means 7 is set to 0 ° and 45 °, respectively. Each peak value is detected and used as comparison coordinates. If the luminance values of these comparison coordinates are equal, the operation is completed, and if they are not equal, the output unit 13 has low illuminance at R, G, B depending on the type of film. It has a function of giving a command to rotate the filter of the light control means 7 by correcting the failure characteristic.
[0024]
When the output unit 13 receives a rotation command for the filter 7a, the output unit 13 sends, for example, a rotation drive signal for rotating the filter in a direction in which the filter changes from a low wavelength side to a long wavelength side and a certain amount of angle to the dimming unit 7. It has a function.
[0025]
Next, the operation of the fluorescence microscope apparatus configured as described above will be described in accordance with the light control control flowchart shown in FIG.
The control device 10 checks the start switch in step # 1, and if this start switch is on, starts the control, and in the next step # 2, the film stored in the external information input means 14 The correction value for correcting the low illuminance failure characteristic in R, G, B is read according to the type.
[0026]
In addition, when there is no designation | designated of a film in the external information input means 14, the correction | amendment of the low illumination intensity failure characteristic in R, G, B is not performed.
Next, in Step # 3, the control device 10 sends a rotational drive signal from the output unit 13 to the light control means 7, and sets the incident angle of the light control means 7 to the filter 7a to be 0 °.
[0027]
When the incident angle to the filter 7a is set to 0 ° in this way, the filter acts as a transmission band on the long wavelength side as shown in FIG.
In this state, the light emitted from the light source 1 is transmitted through the filter 7a of the light control means 7, and the specific wavelength region necessary for exciting the fluorescent dye out of the light emitted from the light source 1 in the dichroic mirror 2. Only the light is reflected as excitation light.
[0028]
The excitation light reflected by the dichroic mirror 2 is condensed on the specimen 4 stained through the objective lens 3.
In the specimen 4, a dye having an excitation wavelength band on the long wavelength side is excited by the excitation light, and fluorescence of that wavelength is generated.
[0029]
The fluorescence from the specimen 4 is captured by the objective lens 3, the wavelength is selectively transmitted by the dichroic mirror 2, further reflected by the quick return mirror 5, and as a specimen image as shown in FIG. 4 by the imaging lens 8. Projected onto the two-dimensional area sensor 9.
[0030]
The two-dimensional area sensor 9 outputs a luminance signal corresponding to the brightness of the sample image projected by the imaging lens 8.
The luminance signal output from the two-dimensional area sensor 9 is stored in the memory unit 11 of the control device 10.
[0031]
The comparison unit 12 reads the luminance signal stored in the memory unit 11, detects the peak value in the luminance signal when the angle of incidence on the filter 7a is 0 °, and uses the coordinates of the peak value as comparison coordinates. .
[0032]
For example, when attention is paid to one of the X lines shown in FIG. 4, the peak value in the luminance signal is point A as shown in FIG. 5, and this is the comparison coordinate.
Next, in step # 4, the control device 10 sends a rotational drive signal from the output unit 13 to the light control means 7, and sets the incident angle of the light control means 7 to the filter 7a to be 45 °.
[0033]
Thus, if the incident angle to the filter 7a is set to 45 °, the filter acts as a transmission band on the low wavelength side as shown in FIG.
In this state, the light emitted from the light source 1 is transmitted through the filter 7a of the light control means 7 as described above, and is necessary for exciting the fluorescent dye out of the light emitted from the light source 1 in the dichroic mirror 2. Only light in a specific wavelength region is reflected as excitation light.
[0034]
The excitation light reflected by the dichroic mirror 2 is collected on the specimen 4 stained by the objective lens 3.
In the specimen 4, a dye having an excitation wavelength band on the low wavelength side is excited by the excitation light, and fluorescence of that wavelength is generated.
[0035]
The fluorescence from the specimen 4 is captured by the objective lens 3, its wavelength is selectively transmitted by the dichroic mirror 2, further reflected by the quick return mirror 5, and the specimen image by the imaging lens 8 on the two-dimensional area sensor 9. Projected.
[0036]
The two-dimensional area sensor 9 outputs a luminance signal corresponding to the brightness of the sample image projected by the imaging lens 8.
The luminance signal output from the two-dimensional area sensor 9 is stored in the memory unit 11 of the control device 10.
[0037]
The comparison unit 12 reads the luminance signal stored in the memory unit 11, detects the peak value in the luminance signal when the incident angle to the filter 7a is 45 °, and uses the coordinates of the peak value as comparison coordinates. .
[0038]
For example, when attention is paid to one of the X lines shown in FIG. 4, the peak value in this luminance signal is point B as shown in FIG. 6, and this is the comparison coordinate.
Next, in step # 5, the comparison unit 12 reads out the respective comparison coordinates when the angles of incidence on the filter 7a are set to 0 ° and 45 °, and compares the luminance values of these comparison coordinates.
[0039]
As a result of the comparison, if the luminance values of the comparison coordinates are equal, the comparison unit 12 ends the work of light control.
However, if the brightness values of these comparison coordinates are not equal, the comparison unit 12 proceeds to step # 6, and the output unit 13 is corrected for low-illuminance failure characteristics in R, G, and B depending on the type of film. To give a command to rotate the filter of the light control means 7.
[0040]
When the output unit 13 receives a rotation command for the filter 7a, the output unit 13 sends, for example, a rotation drive signal for rotating the filter in a direction in which the filter changes from a low wavelength side to a long wavelength side and a certain amount of angle to the dimming unit 7. .
[0041]
Thus, while the filter 7a is rotating, the two-dimensional area sensor 9 outputs a luminance signal corresponding to the brightness of the sample image projected by the imaging lens 8 in step # 7.
[0042]
The luminance signal output from the two-dimensional area sensor 9 is stored in the memory unit 11 of the control device 10. In step # 8, it is determined whether the rotation angle of the filter 7a by the light control means has reached 0 °. If the rotation angle has reached 0 °, the luminance values between the coordinate values determined in steps # 3 and # 4 are compared. If the two luminance values are equal, the adjustment operation is terminated. Move to step # 6. That is, the filter 7a is rotated by the dimming means until the luminance value between the coordinate values determined in steps # 3 and # 4 becomes equal.
[0043]
In this way, dimming control is performed while rotating the filter 7a, and the luminance value between the comparison coordinates determined in steps # 3 and # 4 is not equal, and when the incident angle to the filter 7a returns to 0 °, The comparison unit 12 moves to Step # 9, and filters the luminance value between the comparison coordinates determined in Steps # 3 and # 4 when the incident angle of the filter 7a is 0 ° and 45 ° respectively. 7a is rotated and it complete | finishes.
[0044]
As a result, the fluorescence generated from the specimen 4 is dimmed for each wavelength and has a balanced brightness as shown in FIG.
As described above, in the first embodiment, the brightness is adjusted from the fluorescent image from the sample 4 obtained by the area sensor 9 at 0 ° and 45 ° by the light control means 7 using the characteristics of the filter 7a. After determining the part to be lit, the filter 7a is rotated by the light control means 7 so that the brightness of the part is equal. Therefore, when observing the multiple stained specimen 4, the light control control work is simplified. As a result, the discoloration of the specimen 4 can be reduced, and the brightness of the fluorescence by each fluorescent label can be balanced.
(2) Next, a second embodiment of the present invention will be described. The same parts as those in FIG. 1 are denoted by the same reference numerals, and detailed description thereof is omitted.
[0045]
FIG. 8 is a block diagram of the fluorescence microscope apparatus.
Between the dichroic mirror 2 and the quick return mirror 5, two filters 21 and 22 as the light control means 20 are disposed so as to be detachable with respect to the optical path of the fluorescence from the specimen 4.
[0046]
These filters 21 and 22 are different in transmittance and vary the intensity of fluorescence. For example, one filter 21 has a high transmittance characteristic on the long wavelength side as shown in FIG. The other filter 22 has a transmittance characteristic high on the low wavelength side and low in the other wavelength regions as shown in FIG.
[0047]
The filters 21 and 22 are inserted into and removed from the fluorescent light path in response to a drive signal output from the output unit 13 of the control device 10.
Next, the operation of the fluorescence microscope apparatus configured as described above will be described in accordance with the light control control flowchart shown in FIG.
[0048]
In step # 10, the control device 10 checks the start switch. If the start switch is on, the control device 10 starts control, and the film stored in the external information input means 14 in the next step # 11. The correction value for correcting the low illuminance failure characteristic in R, G, B is read according to the type.
[0049]
In addition, when there is no designation | designated of a film in the external information input means 14, the correction | amendment of the low illumination intensity failure characteristic in R, G, B is not performed.
Next, in Step # 12, the control device 10 sends a drive signal from the output unit 13 to the light control means 20, and inserts one long wavelength filter 21 of the light control means 20 into the fluorescence optical path.
[0050]
In this state, the light emitted from the light source 1 reflects only the light component for a specific wavelength necessary for exciting the fluorescent dye out of the light emitted from the light source 1 in the dichroic mirror 2 as excitation light.
[0051]
The excitation light reflected by the dichroic mirror 2 is collected on the specimen 4 that is multiple-stained by the objective lens 3, and the dye that is dyed is excited in the specimen 4 to generate fluorescence of that wavelength.
[0052]
The fluorescence from the specimen 4 is captured by the objective lens 3, the wavelength is selectively transmitted by the dichroic mirror 2, and only the long wavelength side fluorescence is transmitted by the filter 21, reflected by the quick return mirror 5, and imaged. The image is projected onto the two-dimensional area sensor 9 by the lens 8. The two-dimensional area sensor 9 outputs a luminance signal corresponding to the brightness of the sample image projected by the imaging lens 8.
[0053]
The luminance signal output from the two-dimensional area sensor 9 is stored in the memory unit 11 of the control device 10, where the comparison unit 12 reads the luminance signal stored in the memory unit 11 and inserts the filter 21. The peak value in the luminance signal is detected, and this peak value is used as a comparison coordinate.
[0054]
Next, in Step # 13, the control device 10 sends a drive signal from the output unit 13 to the light control means 7, takes out the filter 21, and inserts the other low-wavelength filter 22 into the fluorescent light path.
[0055]
Thereby, similarly to the above, the two-dimensional area sensor 9 outputs a luminance signal corresponding to the brightness on the low wavelength side in the sample image. This luminance signal is stored in the memory unit 11 of the control device 10.
[0056]
The comparison unit 12 reads the luminance signal stored in the memory unit 11, detects a peak value in the luminance signal when the filter 22 is inserted, and uses the peak value as comparison coordinates.
[0057]
Next, in step # 14, the comparison unit 12 compares the difference in luminance value between comparison coordinates when one filter 21 is inserted with the difference in luminance value between comparison coordinates when the other filter 22 is inserted. Then, based on the result of this comparison, a filter that maximizes the luminance value between the comparison coordinates shall be inserted into the optical path of the fluorescence, and the observed fluorescence is dimmed and balanced for each wavelength. It becomes bright.
[0058]
As described above, in the second embodiment, since the two filters 21 and 22 on the long wavelength side and the low wavelength side are detachably arranged on the optical path of the fluorescence from the specimen 4, the first filter It goes without saying that the same effects as those of the embodiment can be obtained.
(3) Next, a third embodiment of the present invention will be described. The same parts as those in FIG. 1 are denoted by the same reference numerals, and detailed description thereof is omitted.
[0059]
FIG. 12 is a configuration diagram of a fluorescence microscope apparatus corresponding to a triple-stained specimen.
Between the light source 1 and the dichroic mirror 2, first and second light control means 23 and 24 are arranged.
[0060]
These first and second light control means 23 and 24 use filters 23a and 24a whose spectral transmission characteristics change depending on the incident angle of light.
Of these, the filter 23a is rotatably provided so as to be set at an incident angle of 0 ° or 45 ° with respect to the traveling direction of the light from the light source 1, and if the incident angle of the light beam is 0 °, it is shown in FIG. If the non-transmission band is on the long wavelength side as in the characteristic Qa and the incident angle of light is 45 °, the non-transmission band moves to the low wavelength side as in the characteristic Qb shown in FIG. ing.
[0061]
Similarly to the filter 23a, the filter 24a is rotatably provided so as to set the incident angle at 0 ° or 45 ° with respect to the traveling direction of the light from the light source 1, and the incident angle of the light beam is 0 °. If there is, if the transmission band is on the long wavelength side as in the characteristic Pa shown in FIG. 14 and the incident angle of the light beam is 45 °, the transmission band moves to the low wavelength side as in the characteristic Pb shown in FIG. It has transmission characteristics.
[0062]
Next, the operation of the fluorescence microscope apparatus configured as described above will be described in accordance with the light control control flowchart shown in FIG. A sample is shown in FIG.
In step # 20, the control device 10 checks the start switch. If the start switch is turned on, the control device 10 starts control, and the film stored in the external information input means 14 in the next step # 21. The correction value for correcting the low illuminance failure characteristic in R, G, B is read according to the type.
[0063]
In addition, when there is no designation | designated of a film in the external information input means 14, the correction | amendment of the low illumination intensity failure characteristic in R, G, B is not performed.
Next, in step # 22, the control device 10 sends a rotational drive signal from the output unit 13 to the first and second light control means 23, 24, and the first and second light control means 23, 24 are used. The incident angles to the filters 23a and 24a are both set to 0 °.
[0064]
If the incident angles to the filters 23a and 24a are both set to 0 ° in this way, the filter 23a moves to the longer wavelength side as shown by the characteristic Qa in FIG. 13, and the filter 24a As shown by the characteristic Pa of 14, the transmission band moves to the long wavelength side.
[0065]
In this state, the light emitted from the light source 1 is transmitted through the filters 23a and 24a, and the light for a specific wavelength necessary for exciting the fluorescent dye out of the light emitted from the light source 1 in the dichroic mirror 2. Only the components are reflected as excitation light.
[0066]
The excitation light reflected by the dichroic mirror 2 is collected on a specimen 4 that is triple-stained by the objective lens 3, and in this specimen 4, fluorescence due to U excitation is emphasized.
The fluorescence from the specimen 4 is captured by the objective lens 3, its wavelength is selectively transmitted by the dichroic mirror 2, further reflected by the quick return mirror 5, and the specimen image by the imaging lens 8 on the two-dimensional area sensor 9. Projected. FIG. 17 shows the output of the X line on the two-dimensional area sensor.
[0067]
The two-dimensional area sensor 9 outputs a luminance signal corresponding to the brightness of the sample image projected by the imaging lens 8. The luminance signal output from the two-dimensional area sensor 9 is stored in the memory unit 11 of the control device 10.
[0068]
Next, in step # 23, the control device 10 sends a rotational drive signal from the output unit 13 to the first and second light control means 23, 24, and among these, the first light control means 23 is supplied to the filter 23a. The incident angle is set to 45 °, and the incident angle to the filter 24a of the second dimming means 24 is set to 0 °.
[0069]
If the incident angle to each of the filters 23a and 24a is set in this way, the filter 23a moves to the lower wavelength side as shown by the characteristic Qb in FIG. 13, and the filter 24a has the same transmission band as it is. Long wavelength side.
[0070]
In this state, the light emitted from the light source 1 is transmitted through the filters 23a and 24a, and the light for a specific wavelength necessary for exciting the fluorescent dye out of the light emitted from the light source 1 in the dichroic mirror 2. Only the components are reflected as excitation light.
[0071]
The excitation light reflected by the dichroic mirror 2 is collected on a specimen 4 that is triple-stained by the objective lens 3, and in this specimen 4, fluorescence due to B excitation is emphasized.
Similarly to the above, the fluorescence from the specimen 4 is captured by the objective lens 3, its wavelength is selectively transmitted by the dichroic mirror 2, further reflected by the quick return mirror 5, and a two-dimensional area as a specimen image by the imaging lens 8. Projected onto the sensor 9. FIG. 18 shows the output of the X line on the two-dimensional area sensor.
[0072]
Since the two-dimensional area sensor 9 outputs a luminance signal corresponding to the brightness of the sample image projected by the imaging lens 8, the luminance signal is stored in the memory unit 11 of the control device 10.
[0073]
In step # 24, the comparison unit 12 reads out each of the luminance signals stored in the memory unit 11, calculates the difference between these luminance signals, sets the + peak coordinate as the luminance excitation coordinate for U excitation, and the -peak coordinate. Is a luminance comparison coordinate of B excitation. FIG. 19 shows a value obtained by subtracting the luminance value of step # 24 from the luminance value of step # 23.
[0074]
Next, in step # 25, the control device 10 sends a rotational drive signal from the output unit 13 to the first and second light control means 23, 24, and among these, the first light control means 23 is supplied to the filter 23a. The incident angle is set to 0 °, and the incident angle to the filter 24a of the second dimming means 24 is set to 45 °.
[0075]
If the incident angles to the filters 23a and 24a are set in this way, the filter 23a moves the non-transmission band to the long wavelength side as shown by the characteristic Qa in FIG. 13, and the filter 24a has the characteristic Pb in FIG. As shown in FIG. 4, the transmission band moves to the lower wavelength side.
[0076]
In this state, the light emitted from the light source 1 is transmitted through the filters 23a and 24a, and the light for a specific wavelength necessary for exciting the fluorescent dye out of the light emitted from the light source 1 in the dichroic mirror 2. Only the components are reflected as excitation light.
[0077]
The excitation light reflected by the dichroic mirror 2 is collected on a specimen 4 that is triple-stained by the objective lens 3, and fluorescence in the G excitation is emphasized in the specimen 4.
Similarly to the above, the fluorescence from the specimen 4 is captured by the objective lens 3, its wavelength is selectively transmitted by the dichroic mirror 2, further reflected by the quick return mirror 5, and a two-dimensional area as a specimen image by the imaging lens 8. Projected onto the sensor 9.
[0078]
Since the two-dimensional area sensor 9 outputs a luminance signal corresponding to the brightness of the sample image projected by the imaging lens 8, the luminance signal is stored in the memory unit 11 of the control device 10.
[0079]
Next, in Step # 26, the control device 10 sends a rotation drive signal from the output unit 13 to the first and second light control means 23, 24, and the first and second light control means 23, 24 are used. The angles of incidence on the filters 23a and 24a are both set to 45 °.
[0080]
If the incident angles to the filters 23a and 24a are set in this way, the filter 23a moves to the low wavelength side as shown by the characteristic Qb in FIG. 13, and the filter 24a has a low transmission band as it is. On the wavelength side.
[0081]
In this state, the light emitted from the light source 1 is transmitted through the filters 23a and 24a, and the light for a specific wavelength necessary for exciting the fluorescent dye out of the light emitted from the light source 1 in the dichroic mirror 2. Only the components are reflected as excitation light.
[0082]
The excitation light reflected by the dichroic mirror 2 is collected on a specimen 4 that is triple-stained by the objective lens 3, and in this specimen 4, fluorescence due to B excitation is emphasized.
Similarly to the above, the fluorescence from the specimen 4 is captured by the objective lens 3, its wavelength is selectively transmitted by the dichroic mirror 2, further reflected by the quick return mirror 5, and a two-dimensional area as a specimen image by the imaging lens 8. Projected onto the sensor 9.
[0083]
Since the two-dimensional area sensor 9 outputs a luminance signal corresponding to the brightness of the sample image projected by the imaging lens 8, the luminance signal is stored in the memory unit 11 of the control device 10.
[0084]
Next, in step # 27, the comparison unit 12 reads each of the luminance signals stored in the memory unit 11, takes a difference between these luminance signals, and uses the + peak coordinates as luminance comparison coordinates for G excitation.
[0085]
Next, in step # 28, the comparison unit 12 determines the luminance values, the rotation angles of the filters 23 and 24, and the transmission in the three types of luminance comparison coordinates of U, B, and G obtained as described above. Based on the relationship with the rate, an optimum rotation angle at which each fluorescence wavelength can be balanced is calculated.
[0086]
Then, the output unit 13 sends a rotation drive signal having an optimal rotation angle that can be balanced at each fluorescence wavelength calculated by the comparison unit 12 to each of the filters 23a and 24a.
[0087]
Thereby, each filter 23a, 24a rotates and can balance each fluorescence in the sample 4 dye | stained in triple.
As described above, in the third embodiment, since the first and second dimming means 23 and 24 are arranged between the light source 1 and the dichroic mirror 2, the sample 4 dyed three times is observed. In this case, the dimming control work can be simplified, the fading of the specimen 4 can be reduced, the brightness of the fluorescence by the three fluorescent labels can be balanced, and peak values other than the target measurement wavelength can be obtained. Can be removed.
The present invention is not limited to the first to third embodiments, and various modifications may be made.
[0088]
【The invention's effect】
As described in detail above, according to the first to third aspects of the present invention, in order to observe a multiple-stained specimen, the work is simplified and the fading of the specimen is reduced, and the fluorescence of each fluorescent label is reduced. It is possible to provide a fluorescence microscope apparatus that can balance brightness.
According to the third aspect of the present invention, a fluorescence microscope apparatus can be provided that can balance the brightness of each fluorescence of a sample that has been triple-stained.
[Brief description of the drawings]
FIG. 1 is a configuration diagram showing a first embodiment of a fluorescence microscope apparatus according to the present invention.
FIG. 2 is a characteristic diagram of a filter used as dimming means.
FIG. 3 is a flowchart of light control in the fluorescence microscope apparatus.
FIG. 4 is a schematic diagram showing a sample image projected on a two-dimensional area sensor.
FIG. 5 is a diagram showing a peak value when excited on a long wavelength side.
FIG. 6 is a diagram showing a peak value when excited on a low wavelength side.
FIG. 7 is a diagram showing the brightness of fluorescence from a dimmed specimen.
FIG. 8 is a configuration diagram showing a second embodiment of the fluorescence microscope apparatus according to the present invention.
FIG. 9 is a characteristic diagram of one filter used as dimming means.
FIG. 10 is a characteristic diagram of the other filter used as the dimming means.
FIG. 11 is a flowchart of light control in the fluorescence microscope apparatus.
FIG. 12 is a configuration diagram showing a third embodiment of a fluorescence microscope apparatus according to the present invention.
FIG. 13 is a characteristic diagram of a filter in the first dimming unit.
FIG. 14 is a characteristic diagram of a filter in the second dimming means.
FIG. 15 is a flowchart of light control in the fluorescence microscope apparatus.
FIG. 16 shows a sample.
FIG. 17 is a diagram showing an output on an X line on a two-dimensional line sensor.
FIG. 18 is a diagram showing an output on an X line on a two-dimensional line sensor.
FIG. 19 is a diagram showing a value of a difference in luminance values between comparative coordinates.
[Explanation of symbols]
1 ... light source,
2 ... Dichroic mirror,
3 ... Objective lens,
4 ... Sample,
5 ... Quick return mirror,
6 ... Camera,
7: Light control means,
7a ... filter,
8 ... imaging lens,
9 ... 2D area sensor,
10 ... Control device,
11 ... Memory part,
12 ... Comparison part,
13 ... output part,
14 ... External information input means,
20: Light control means,
21, 22 ... filter,
23 ... 1st light control means,
24 ... Second light control means,
23a, 24a ... filters.

Claims (3)

In a fluorescence microscope apparatus that irradiates excitation light to a multiple fluorescently labeled specimen and observes the fluorescence emitted from the specimen,
A detection element for detecting the brightness of the fluorescence emitted from the specimen;
A dimming means for dimming the fluorescence emitted from the specimen for each wavelength;
Control means for controlling the light control means so that the brightness of the fluorescence observed based on the brightness of the fluorescence for each wavelength detected by the detection element becomes equal for each wavelength. A fluorescence microscope apparatus. 2. The fluorescence microscope apparatus according to claim 1, wherein the light control means is a filter whose spectral transmission characteristics change depending on the incident angle of light. 2. The fluorescence microscope apparatus according to claim 1, wherein the light control means is a filter having different transmittance in a specific wavelength region.

Images