JPH08320292A - Device and method for observing fluorescence - Google Patents

Abstract

PURPOSE: To provide a method and a device for observing fluorescence in which a highly precise fluorescence observation can be realized. CONSTITUTION: The fluorescence from a specimen 13 excited by laser beam is separated into a long wavelength one and a short wavelength one through a dichroic mirror 14, and they are passed through absorption filters 15, 17, and detected as data by short wavelength and long wavelength detecting detectors 16, 18. The fluorescence data related to long wavelength and short wavelength detected by arranging the absorption filters 15, 17 in the respective detecting optical paths of the detectors 16, 18 and the leak fluorescence data of short wavelength on the long wavelength side detected by arranging the absorption filter 15 used for short wavelength detection on the long wavelength detecting detector 18 are stored in an image memory, respectively, and the differential result of these stored fluorescence data is stored in the image memory as long wavelength-side corrected image data, and also displayed on an image monitor.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fluorescence observation method and apparatus used for observing multiple stained specimens.

[0002]

2. Description of the Related Art Conventionally, when observing a double-stained specimen using a scanning laser microscope, two kinds of fluorescence excited by a laser beam are converted into a long-wavelength one and a short-wavelength one through a dichroic mirror. The data is separated and passed through an absorption filter, and then detected as data by a detector.

However, in general, a dichroic mirror cannot completely reduce the transmittance of short wavelengths to 0 because of its characteristics, so that two kinds of fluorescence can be converted into a long wavelength one and a short wavelength one by only using a dichroic mirror. It is difficult to separate, and short wavelength light may leak to the long wavelength side.

Therefore, conventionally, by narrowing the band of the long-wavelength detection side filter, the leakage light of the short-wavelength fluorescence entering the detector is removed, or the long-wavelength side fluorescence detection data and the short-wavelength side fluorescence are detected. By comparing the detection data of, the data detected on the short-wavelength side and detected as weak data on the long-wavelength side is regarded as short-wavelength leaked light, and a method of removing this portion from the long-wavelength side, etc. Is being considered.

[0005]

However, in the previous method of narrowing the band of the absorption filter on the long wavelength detection side, the narrower the band of the filter is, the weaker the light passing therethrough becomes. There is a problem that the S / N ratio of the fluorescence on the side is deteriorated and the cost for producing such an absorption filter is high.

Further, in the method of comparing the detection data on the long wavelength side with the detection data on the short wavelength side, the weak data on the long wavelength side is the original weak fluorescence on the long wavelength side or the short fluorescence. Since it is not possible to strictly determine whether the light has a wavelength leak, there is a problem that the detection data on the long wavelength side becomes inaccurate as a result. The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a fluorescence observation method and apparatus capable of realizing highly accurate fluorescence observation.

[0007]

According to the first aspect of the present invention,
In the fluorescence observation method in which the fluorescence excited by the laser light is separated into long-wavelength and short-wavelength ones through a dichroic mirror, and each is detected as data through an absorption filter, in the short-wavelength detection side and the long-wavelength detection side To detect the fluorescence data on the long wavelength and the short wavelength, and to detect the short wavelength leakage fluorescence data on the long wavelength side by arranging the absorption filter used for the short wavelength detection on the long wavelength detection side. Then, the corrected data on the long wavelength side is obtained from the difference between the detected fluorescence data.

According to the second aspect of the present invention, the fluorescence excited by the laser beam is separated into a long wavelength and a short wavelength through a dichroic mirror and detected as data by a long wavelength and short wavelength detector through an absorption filter. In the fluorescence observation device, first storage means for storing fluorescence data relating to the long wavelength and the short wavelength detected by disposing an absorption filter in the detection optical path of each of the long wavelength and short wavelength detectors; Second storage means for arranging the absorption filter used for the short wavelength detection on the wavelength detector side to store the leaked fluorescence data of the short wavelength on the long wavelength side detected, and these first and second storage means And a control unit for outputting corrected data on the long wavelength side from the difference between the fluorescence data stored in.

According to a third aspect of the invention, in the second aspect, the absorption filter is provided so as to be freely inserted into and removed from the detection optical path of each of the long wavelength detector and the short wavelength detector.

[0010]

As a result, according to the invention of claim 1, the fluorescence data concerning the long wavelength and the short wavelength detected by disposing the absorption filters on the short wavelength detection side and the long wavelength detection side, respectively, and the fluorescence data Since the absorption filter used for the short wavelength detection is arranged on the wavelength detection side to obtain the difference with the short wavelength leakage fluorescence data on the long wavelength side to be detected, the short wavelength fluorescence leaking to the long wavelength side is detected. It is possible to obtain pure fluorescence data on the long wavelength side with the fraction removed.

According to the second aspect of the invention, fluorescence data relating to the long wavelength and the short wavelength detected by disposing an absorption filter in the detection optical paths of the long wavelength and short wavelength detectors respectively, and Short-wavelength leaked fluorescence data on the long-wavelength side detected by arranging the absorption filter used for the short-wavelength detection on the long-wavelength detector side is stored in the storage means respectively, and the difference is stored from the stored fluorescence data. Since the corrected data on the wavelength side is obtained, pure fluorescence data on the long wavelength side can be obtained by removing the short wavelength fluorescence component leaking to the long wavelength side.

According to the third aspect of the invention, since the absorption filter can be freely inserted into and removed from the detection optical paths of the long-wavelength detector and the short-wavelength detector, the operation for fluorescence observation is simple. You can

[0013]

Embodiments of the present invention will be described below with reference to the drawings. (First Embodiment) FIG. 1 shows a schematic structure of a fluorescence observation apparatus to which the present invention is applied. In the figure, reference numeral 1 is a controller. The controller 1 is connected to an operation panel 2, a fluorescence detection unit 3, and an image memory 4, an image monitor 5 is connected to the image memory 4, and the controller 1 is instructed by the operation panel 2. A fluorescence detection start signal is output from the fluorescence detection unit 3 to the detection output from the fluorescence detection unit 3 as image data to the image memory 4 and an observation image is displayed on the image monitor 5.

FIG. 2 shows a schematic structure of the fluorescence detecting section 3. In this case, the laser light oscillated from the laser oscillator 10 is passed through the excitation light selection filter 11 to generate desired excitation light, the excitation light is scanned on the sample 13 via the dichroic mirror 12, and emitted from the sample 13. The fluorescent light passes through the dichroic mirror 12 and enters the dichroic mirror 14. Then, the short wavelength side is reflected by the dichroic mirror 14 and converted into data by the short wavelength detection detector 16 through the absorption filter 15, while the long wavelength side is transmitted through the dichroic mirror 14 and passed through the absorption filter 17. Data is converted by the detector 18 for wavelength detection.

A broken line 109 in the figure shows how the short-wavelength leaked light from the dichroic mirror 14 is input to the long-wavelength detector 18 through the absorption filter 17, as described above.

However, in such a configuration, as shown in FIG.
The operation according to the flowchart shown in is executed. First, the observer sets absorption filters 15 and 17 on the short wavelength detection detector 16 side and the long wavelength detection detector 18 side, respectively, in step 301, and in step 302 sets various scanning conditions on the operation panel 2. To do.
Then, a fluorescence detection start signal is output from the controller 1 to the fluorescence detection unit 3, and the laser light oscillated from the laser oscillator 10 passes through the excitation light selection filter 11 to become the desired excitation light and the sample 13 via the dichroic mirror 12. Scanned above. Then, the fluorescence from the sample 13 passes through the dichroic mirror 12 and the dichroic mirror 14
When incident on the short wavelength side, the one on the short wavelength side is reflected and converted into data by the detector 16 for detecting the short wavelength through the absorption filter 15, and the one on the long wavelength side is transmitted through the absorption filter 17 for detecting the long wavelength. It is converted into data by the detector 18 of.

In this way, long-wavelength and short-wavelength data are fetched, and when one screen of data is fetched, the controller 1 instructs the step 303 to change the long-wavelength and short-wavelength data. Each data A is image memory 4
Stored in.

Next, in step 304, the observer removes the absorption filter 17 on the long wavelength detecting detector 18 side and replaces it with the absorption filter 15 on the short wavelength detecting detector 16 side.

Then, in step 305, when the fluorescence detection start signal is output from the controller 1 to the fluorescence detection section 3 in the same manner as described above, the detector 18 for long wavelength detection is output.
The image data of only the long wavelength, which has been converted into data in step 1, is taken in as the image data of the leakage fluorescence of the short wavelength to the long wavelength side. Then, in accordance with an instruction from the controller 1, step 3
At 06, the data B is stored in the image memory 4. In this case, the storage of the image data in the image memory 4 is set in an area different from the storage location of each of the long-wavelength and short-wavelength image data obtained in step 303 described above.

Next, in step 307, the controller 1 subtracts the short wavelength leak fluorescence data B to the long wavelength side obtained in step 306 from the data A obtained in step 303, and the result is subtracted from the long wavelength. The image data is stored in the image memory 4 as the corrected image data on the side, and in step 308, the long-wavelength and short-wavelength image data are displayed on the image monitor 5.

Therefore, in this way, the fluorescence from the sample 13 excited by the laser light is separated into the long wavelength and the short wavelength through the dichroic mirror 14, and the short wavelength and the long wavelength are separated through the absorption filters 15 and 17, respectively. The detectors 16 and 18 for wavelength detection detect the data, and the absorption filters 15 and 17 are arranged in the detection optical paths of the detectors 16 and 18 for short wavelength detection and long wavelength detection, respectively. The long-wavelength and short-wavelength fluorescence data are stored in the image memory 4, and the long-wavelength side detected by arranging the absorption filter 15 used for the short-wavelength detection on the long-wavelength detector 18 side. The short-wavelength leakage fluorescence data from the image memory 4
Stored in the image memory 4, the difference between the fluorescence data previously stored in the image memory 4 and this data is obtained, and the result is stored in the image memory 4 as corrected image data on the long-wavelength side. Since each image data of the above is displayed on the image monitor 5, it is possible to obtain pure long-wavelength fluorescence data from which short-wavelength fluorescence components leaking to the long-wavelength side are removed. Observation will be realized. (Second Embodiment) FIG. 4 shows a specific example of the switching configuration of the absorption filters 15 and 17, in which a filter slider 19 is provided between the dichroic mirror 14 and the short wavelength detector 16. Along with the arrangement, the dichroic mirror 14 and the detector 18 for detecting long wavelengths
The filter slider 20 is also disposed between and.

These filter sliders 19 and 20 are
The absorption filters 15 and 17 can be housed respectively, and these absorption filters 15 and 17 can be selectively taken in and out of the detection optical path.

However, in such a structure, first, as shown in the figure, the absorption filter 1 is attached to the filter slider 19.
5. The absorption filter 17 is set on the filter slider 20 respectively, and in this state, the long wavelength and short wavelength data are taken in. Next, the absorption filter 17 of the filter slider 20 is removed, and the filter slider 19 is removed. By replacing the absorption filter 15 on the filter slider 20 side and capturing the image data of the leakage fluorescence of the short wavelength to the long wavelength side, the corrected image data of the long wavelength side can be obtained as described above. become.

In FIG. 4, the absorption filter 15 for detecting the short wavelength is pulled out from the filter slider 19 and put in the filter slider 20, but another absorption filter 15 is prepared. , It is also possible to detect short-wavelength leakage fluorescence.

Therefore, in this way, by adopting the filter sliders 19 and 20, the absorption filter 1
Since 5 and 17 can be easily put in and taken out, the operation for fluorescence observation can be made simple. (Third Embodiment) FIGS. 5A and 5B show an absorption filter 1
5 and 17 show other specific examples of the switching configuration,
In this case, the mirror 21 is arranged in the reflection optical path of the dichroic mirror 14, the detection optical path of the short wavelength detection detector 16 is bent at a right angle, and the long wavelength detection detector 18 is formed.
Forming a detection optical path parallel to the detection optical path.

Then, these short wavelength detectors 1
The turrets 22 and 23 with absorption filters are arranged in the respective detection optical paths of the detector 6 and the detector 18 for long wavelength detection.

These turrets 22 with absorption filters,
23, absorption filters 15 and 17 are separately provided as shown in FIG.
By rotating the absorption filters 15 and 1
7 can be put in and taken out from each detection optical path.

However, in such a structure, first, the turrets 22 and 23 are rotated to install the absorption filters 15 and 17 in the detection optical paths of the short-wavelength detector 16 and the long-wavelength detector 18, respectively. Separately, each data of long wavelength and short wavelength is taken in, and then the turrets 22 and 23 are rotated again to remove the absorption filter 17 in the detection optical path of the detector 18 for long wavelength detection. By setting the absorption filter 15 in the detection optical path of the long-wavelength detector 18 and capturing the image data of the short-wavelength leakage fluorescence to the long-wavelength side,
As described above, corrected image data on the long wavelength side can be obtained.

Accordingly, by adopting the turrets 22 and 23 with absorption filters, the absorption filters 15 and 17 can be easily taken in and out, and in this case also, the operation for fluorescence observation is performed. Can be done easily.

In principle, the absorption filters 15 and 17 in each of the above-described embodiments are put in and taken out by an observer, but for example, the turrets 22 and 23 with absorption filters of the third embodiment are used. If the above-described rotation operation of the turrets 22 and 23 is controlled based on the instruction of the controller 1, the corrected image data on the long wavelength side can be obtained fully automatically. .

In each of the above-described embodiments, first, the absorption filters 15 and 17 are provided in the detection optical paths of the short wavelength detection detector 16 and the long wavelength detection detector 18, respectively.
Is set to capture each data of long wavelength and short wavelength, and then the absorption filter 17 in the detection optical path of the detector 18 for long wavelength detection is removed and the absorption filter 15 is set in the detection optical path of the detector 18 for long wavelength detection. Then, the operation of taking in the image data of the short-wavelength leakage fluorescence to the long-wavelength side is alternately executed, but, for example, while moving the stage and continuously correcting the long-wavelength side, In the case of retrieving image data repeatedly, first, retrieve the first image data in the order described above, and then
When the stage is moved and the second data is taken in, the setting state of the absorption filter is left unchanged, and the image data of the leakage fluorescence of the short wavelength to the long wavelength side is taken in first.
Next, the absorption filters 15 and 17 are returned to the detection optical paths of the normal short-wavelength detection detector 16 and the long-wavelength detection detector 18, respectively, and long-wavelength and short-wavelength image data are taken in to obtain long-wavelength detection. By obtaining the corrected image data on the side and repeating such an operation, the set number of images can be captured.

In this way, efficient fluorescence observation can be realized when a plurality of pieces of image data are continuously captured. Although the invention has been described above based on the embodiment, the invention includes the following inventions.

(1) In a fluorescence observation method in which fluorescence excited by a laser beam is separated into a long wavelength and a short wavelength through a dichroic mirror, and each is detected as data through an absorption filter, a short wavelength detection side and a long wavelength are detected. An absorption filter is arranged on the detection side to detect fluorescence data on the long wavelength and the short wavelength,
The short wavelength leak fluorescence data on the long wavelength side is detected by arranging the absorption filter used for the short wavelength detection on the long wavelength detection side, and the long wavelength side is corrected from the difference between these detected fluorescence data. A fluorescence observation method characterized by obtaining data.

With this configuration, the fluorescence data on the long wavelength and the short wavelength detected by disposing the absorption filters on the short wavelength detection side and the long wavelength detection side, respectively, and the short wavelength on the long wavelength detection side. The absorption filter used for detection is arranged to obtain the difference with the short-wavelength leaked fluorescence data on the long-wavelength side to be detected, so the short-wavelength fluorescent light leaking to the long-wavelength side is removed, Fluorescence data on the long wavelength side can be obtained, and highly accurate fluorescence observation can be realized.

(2) In the fluorescence observation apparatus, the fluorescence excited by the laser beam is separated into long-wavelength and short-wavelength ones through a dichroic mirror, and is detected as data by a long-wavelength and short-wavelength detector through an absorption filter, respectively. First storage means for arranging an absorption filter in each of the detection optical paths of the long-wavelength and short-wavelength detectors to store fluorescence data relating to the detected long-wavelengths and short-wavelengths, and to the long-wavelength detector side. Second storage means for storing short-wavelength leaked fluorescence data on the long-wavelength side, which is detected by arranging an absorption filter used for short-wavelength detection, and fluorescence stored in these first and second storage means A fluorescence observation apparatus comprising: a control unit that outputs corrected data on the long wavelength side from the difference between the data.

In this way, the fluorescence data on the long wavelength and the short wavelength detected by disposing the absorption filters in the detection optical paths of the long wavelength and short wavelength detectors, and the fluorescence data on the long wavelength detector side are detected. Short-wavelength leakage fluorescence data on the long-wavelength side detected by arranging the absorption filter used for short-wavelength detection is stored in the storage means, respectively, and the long-wavelength side is corrected from the difference between the stored fluorescence data. Since data is obtained, pure fluorescence data on the long wavelength side can be obtained in this case as well, with the short wavelength fluorescence component leaking to the long wavelength side removed, and highly accurate fluorescence observation can be realized.

(3) In the fluorescence observation apparatus described in (2), the absorption filter is provided so as to be freely inserted into and removed from the detection optical path of each of the long wavelength detector and the short wavelength detector.

With this arrangement, the absorption filter can be freely inserted into and removed from the detection optical paths of the long-wavelength detector and the short-wavelength detector, so that the operation for fluorescence observation can be simplified.

[0039]

As described above, according to the present invention, it is possible to obtain pure long-wavelength fluorescence data from which short-wavelength fluorescence components leaking to the long-wavelength side are removed, and thereby highly accurate. Fluorescence observation can be realized.

[Brief description of drawings]

FIG. 1 is a diagram showing a schematic configuration of a first embodiment of the present invention.

FIG. 2 is a diagram showing a schematic configuration of a fluorescence detection unit.

FIG. 3 is a flowchart for explaining the operation of the first embodiment.

FIG. 4 is a diagram showing a switching configuration of an absorption filter according to a second embodiment of the present invention.

FIG. 5 is a diagram showing a switching configuration of an absorption filter according to a third embodiment of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 ... Controller, 2 ... Operation panel, 3 ... Fluorescence detection part, 4 ... Image memory, 5 ... Image monitor, 10 ... Laser oscillator, 11 ... Excitation light selection filter, 12 ... Dichroic mirror, 13 ... Specimen, 14 ... Dichroic mirror , 15 ... Absorption filter, 16 ... Detector for detecting short wavelength, 17 ... Absorption filter, 18 ... Detector for long wavelength detection, 19, 20 ... Filter slider, 21 ... Mirror, 22, 23 ... Turret with absorption filter.

Claims (3)

[Claims] 1. A fluorescence observation method in which fluorescence excited by laser light is separated into long-wavelength and short-wavelength ones through a dichroic mirror, and each is detected as data through an absorption filter. Short-wavelength detection side and long-wavelength detection Detect fluorescence data on the long wavelength and the short wavelength by arranging an absorption filter on each side, and arrange the absorption filter used for the short wavelength detection on the long wavelength detection side to detect the short wavelength on the long wavelength side. A method for observing fluorescence, characterized by detecting leaked fluorescence data and obtaining corrected data on the long wavelength side from the difference between the detected fluorescence data. 2. A fluorescence observation apparatus which separates fluorescence excited by laser light into long-wavelength and short-wavelength ones through a dichroic mirror, and detects the data as data by a long-wavelength and short-wavelength detector through an absorption filter, respectively. First storage means for arranging an absorption filter in each of the detection optical paths of the long-wavelength and short-wavelength detectors to store fluorescence data relating to the detected long-wavelengths and short-wavelengths; and the short-circuit on the long-wavelength detector side. Second storage means for storing short-wavelength leakage fluorescence data on the long-wavelength side, which is detected by arranging an absorption filter used for wavelength detection, and fluorescence data stored in these first and second storage means And a control means for outputting corrected data on the long wavelength side from the difference between the fluorescence observation apparatus. 3. The fluorescence observation apparatus according to claim 2, wherein the absorption filter is provided so as to be freely inserted into and removed from the detection optical paths of the long-wavelength detector and the short-wavelength detector, respectively.