JP3413967B2 - Fluorescence microscope - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fluorescence microscope. More specifically, the present invention relates to an optical arrangement of a laser scanning fluorescence microscope for irradiating a sample labeled with a plurality of fluorescent dyes with laser light of a plurality of wavelengths to observe the sample. 2. Description of the Related Art The optical arrangement of a conventional laser scanning microscope is as shown in FIG. Laser head 1,
The optical heads 2 and 3 and the dichroic mirrors 4 and 5 form an optical system for excitation light.
The laser beams L1, L2, L3 emitted from 3 are reflected by the dichroic mirror 6 and introduced into the optical path for irradiating the sample 11 via the scanning unit 7, the relay lenses 8, 9 and the objective lens 10. Fluorescence F1, F2, F3 is emitted from the sample 11 by being excited by the irradiation of the laser beams L1, L2, L3. The photodetectors 12, 13, 14 and the dichroic mirrors 15, 16 form an optical system for fluorescence,
The fluorescent light F1, F2, F3 emitted from the specimen 11 travels backward and passes through the dichroic mirror 6, and each of the light detectors 1
Light is incident on 2, 13, and 14. Since the light travels as described above, the dichroic mirror 4 reflects the laser light L1 and
2, the dichroic mirror 5 reflects the laser light L2 and transmits the laser light L3. The dichroic mirror 15 reflects the fluorescent light F1, transmits the fluorescent light F2 and F3, the dichroic mirror 16 reflects the fluorescent light F2,
Transmits the fluorescent light F3. The dichroic mirror 6 reflects all of the laser beams L1, L2, and L3, and
Both 2 and F3 are transmitted. [0004] The spectral characteristics of the laser dichroic mirrors 4 and 5 and the fluorescent dichroic mirrors 15 and 16 may be any spectral characteristics that reflect the excitation light side and transmit the fluorescent light side at a predetermined wavelength. However, the spectral characteristics of the dichroic mirror 6 are more complicated. An example of the spectral transmission characteristics of the dichroic mirror 6 will be described with reference to FIG. FIG. 7 shows the wavelength on the horizontal axis and the transmittance on the vertical axis, and all the light that does not pass is reflected. The dichroic mirror 6 reflects a plurality of discontinuous bands of light,
It has a spectral characteristic of transmitting light of a wavelength in a band between those bands. A, B, and C indicate the wavelengths of the minimum transmittance, that is, the maximum reflectance, and λA, λB, and λC indicate the laser light L, respectively.
1, L2 and L3, and λa, λb and λc are the wavelengths of the fluorescent light F1, F2 and F3, respectively. λA, λB,
.lambda.c substantially coincides with the reflectance maxima A, B, and C, respectively.
λa, λb, and λc correspond to portions having higher transmittance on the longer wavelength side than A, B, and C, respectively. Generally, the wavelength of fluorescence is not a wavelength that is significantly different from the wavelength of excitation light. Thus, λa is longer than λA but shorter than λB, and similarly λb is longer than λB but shorter than λC. [0006] As described above, the conventional fluorescence microscope has a spectral characteristic that reflects light in a plurality of discontinuous bands and transmits light in a wavelength band between the bands. The optical system for excitation light and the optical system for fluorescence are combined by the dichroic mirror provided, and the single dichroic mirror reflects the excitation light, transmits the fluorescence, and separates the two. [0007] By the way, the fluorescence image of a specimen observed with a fluorescence microscope is often very dark, and therefore, it has been a major problem how to efficiently form fluorescence. In order to obtain a bright fluorescent image, much light other than the excitation light must be condensed on the image plane. There is a limit in improving the transmittance of the objective lens and the relay lens, and efforts are being made to improve the spectral transmittance characteristics of the dichroic mirror. Good spectral transmittance characteristics are high reflectance of excitation light and high transmittance of fluorescence, but usable laser light wavelengths are limited to a small number of specific wavelengths, and the wavelength band of laser light Is a few nm
There is a very narrow feature. Fluorescence has spectral characteristics in a broad wavelength band which is close to the excitation wavelength and slightly wider on the long wavelength side by about 10 to 30 nm therefrom. A dichroic mirror used when a single laser beam is excitation light reflects almost all wavelengths shorter than the wavelength of the laser beam and transmits almost all light having a wavelength longer than the wavelength of the laser beam. It was sufficient to have simple characteristics, and it was easy to manufacture and inexpensive. When a plurality of laser lights are excitation lights,
There is a need for a dichroic mirror that has a plurality of reflection maxima that match the wavelength band of laser light limited to a plurality of specific discontinuous wavelengths and has high transmittance in other wavelength bands. For this purpose, a multi-layer coating film having a large number of coating layers must be used. However, it is not possible to sufficiently match the maximum reflection wavelength with the wavelength of the laser beam, and as shown in FIG.
And the coincidence of λB becomes worse,
Also, the width of the reflection band was unnecessarily widened. Even if the number of coating layers is increased and design efforts are made, the design and manufacture of such a multi-layer coding film is not easy, and the characteristics vary widely, the yield is poor and the cost is very high. There was a problem that it was difficult to supply at a low price. SUMMARY OF THE INVENTION In view of the above problems, the present invention relates to a method in which a plurality of excitation lights having different wavelengths emitted from a plurality of light sources and a plurality of fluorescent lights having different wavelengths emitted from a sample excited by irradiation of the excitation lights are provided. To provide a fluorescence microscope capable of easily obtaining a bright fluorescent image that can be separated only by a wavelength separator having a simple spectral characteristic such that incident light is separated into reflected light and transmitted light at a wavelength. Aim. According to the present invention, an excitation light having a first wavelength is generated, and a sample is irradiated to generate fluorescence having a second wavelength longer than the first wavelength. A first light source and a third light source for generating excitation light having a third wavelength longer than the second wavelength and irradiating the sample to generate fluorescence having a fourth wavelength longer than the third wavelength. A second light source, first light separating means having a spectral characteristic of separating light at a wavelength set between the first wavelength and the second wavelength, and the third wavelength and A second light separating unit having a spectral characteristic for separating light at a wavelength set between the fourth wavelength and a wavelength set between the second wavelength and the third wavelength; The light source has a spectral characteristic that separates light at the boundary, and the excitation light from the first light source and the excitation light from the second light source are A third light separating means for guiding light from the sample according to the spectral characteristics while guiding the light to the same optical path to the sample, and one of the lights from the sample separated by the third light separating means. The first light detected through the first light separating means
And a second light detecting means for detecting, through the second light separating means, the other of the light from the sample, separated by the third light separating means. . The pumping light of the first wavelength from the first light source is the first excitation light.
The excitation light of the second wavelength from the second light source is separated by the second light separating means, and then guided to the same optical path by the third light separating means to sample the sample. Irradiate. The fluorescence of the second wavelength and the fluorescence of the fourth wavelength from the specimen are separated by the third light separating means. The fluorescence of the second wavelength is separated by the first light separation means and detected by the first light detection means, and the fluorescence of the fourth wavelength is separated by the second light separation means and Is detected by the light detecting means. An embodiment of the present invention will be described with reference to FIG. FIG. 1 shows the optical arrangement of the laser scanning microscope. The laser head 21 and the laser head 22 each have a wavelength of 488.
nm laser light L4 and 568 nm wavelength laser light L5
Is a light source that emits light. The photodetector 23 is a photoelectric element that detects the fluorescence F4 emitted from the sample 11 by the irradiation of the laser light L4, and the photodetector 24 is a photoelectric element that detects the fluorescence F5 emitted from the sample 11 by the irradiation of the laser light L5. . The dichroic mirror 25 is a wavelength separator that reflects the laser light L4 and transmits the fluorescent light F4, and the dichroic mirror 26 reflects the laser light L5 and transmits the fluorescent light F5. The laser head 21, the photodetector 23, and the dichroic mirror 25, and the laser head 22, the photodetector 24, and the dichroic mirror 26 form a set. The scanning unit 7, the relay lenses 8, 9 and the objective lens 10 form an objective lens optical system for converging and irradiating the sample 11 with the laser light L4 and the laser light L5. Head 22, photodetector 2
3 and the photodetector 24 are the sample 11 by the objective lens optical system.
Are arranged on the image plane. The dichroic mirror 27 has a laser beam L4
And a wavelength separator that reflects the fluorescent light F4 and transmits the laser light L5 and the fluorescent light F5.
The optical path from the dichroic mirror 25 and the dichroic mirror 26 is connected to the optical path of the objective lens optical system through the optical path. Next, a laser beam L4 for irradiating the specimen 11 labeled with a fluorescent substance and a fluorescence F4 for exciting and emitting light,
The spectral distribution of the laser light L5 and the fluorescence F5 that emits light when excited will be described with reference to FIG. 2 using the sample 11 labeled with the fluorescent substance fluorescein and the fluorescent substance rhodamine as an example. FIG. 2 shows the wavelength on the horizontal axis and the intensity factor on the vertical axis. The fluorescent substance fluorescein has a wavelength of 488 n.
m is excited by the laser light L4, and emits a fluorescent light F4 having a maximum value at a wavelength of about 520 nm. . Although the half widths of the laser light L4 and the laser light L5 are small, the fluorescence F4 and the fluorescence F5 are broad and the half widths are large. Next, the spectral characteristics of the dichroic mirror 25, the dichroic mirror 26, and the dichroic mirror 27 will be described. FIG. 3 shows the wavelength on the horizontal axis and the transmittance on the vertical axis, and all the light that does not pass is reflected. The dichroic mirror 25 and the dichroic mirror 26 have spectral characteristics of transmitting light having wavelengths of 488 nm and 568 nm, respectively, and reflecting light exceeding the wavelengths. The rise of the spectral transmittance curve of the dichroic mirror 25 is 48
It is set to the nearest long wavelength side of 8 nm. In order to reflect the laser light L4 and transmit the fluorescence F4, it is necessary to set the rise of the spectral transmittance curve between 488 nm and 520 nm, and furthermore, although the half width of the laser light L4 is small, This is because the fluorescence F4 is broad and has a large half width. The rise of the spectral transmittance curve of the dichroic mirror 26 is set to the nearest long wavelength side of 568 nm for the same reason. The rise of the spectral transmittance curve of the dichroic mirror 27 is set at around 560 nm. This reflects the laser light L4 and the fluorescent light F4, and the laser light L5
In order to transmit the fluorescence F5, it is necessary to set the rise of the spectral transmittance curve between 520 nm and 568 nm, and further, although the half width of the laser light L5 is small,
This is because the fluorescence F4 is broad and has a large half width. As described above, the dichroic mirror 27 only needs to be set so that the rising edge is around 560 nm. Therefore, the dichroic mirror 27 can be easily and inexpensively manufactured because it has a very simple spectral characteristic that reflects all the light in the band below the rising edge and transmits all the light in the band beyond the rising edge. Next, the operation will be described. The laser light L4 emitted from the laser head 21 is reflected by the dichroic mirror 25 and then by the dichroic mirror 27, and is introduced into the objective lens optical system.
The laser light L5 emitted from 2 is reflected by the dichroic mirror 26, passes through the dichroic mirror 27, and is introduced into the objective lens optical system. The laser light L4 and the laser light L5 are condensed by the objective lens 10 via the scanning unit 7, the relay lenses 8 and 9, and irradiate the sample 11. Fluorescent light F4 and fluorescent light F5 are emitted from the sample 11, and converged by the objective lens 10, and then travel backward through the relay lenses 8, 9 and the scanning unit 7. Fluorescent F4
Is reflected by the dichroic mirror 27, passes through the dichroic mirror 25, and enters the photodetector 23, and the fluorescent light F5 passes through the dichroic mirror 27 and the dichroic mirror 26, enters the photodetector 24, and a fluorescent image is formed. can get. A second embodiment of the present invention will be described with reference to FIG. A detailed description of the same or similar points as in the above-described embodiment will be omitted. The laser head 21, the laser head 22, and the laser head 29 are respectively a laser beam L4 having a wavelength of 488 nm, a laser beam L5 having a wavelength of 568 nm, and a laser beam 647n.
It is a light source that emits m laser light L6. Photodetector 23
Is a photoelectric element for detecting fluorescence F4 emitted from the sample 11 by irradiation of the laser light L4, and the photodetector 24 is a laser light L5
And the photodetector 30 is a photoelectric element that detects the fluorescent light F6 emitted from the sample 11 by the irradiation of the laser beam L6. The dichroic mirror 25 reflects the laser light L4 and transmits the fluorescent light F4, the dichroic mirror 26 reflects the laser light L5 and transmits the fluorescent light F5, and the dichroic mirror 31 reflects the laser light L6 and transmits the fluorescent light F6. It is a wavelength separator. The scanning unit 7, the relay lenses 8, 9 and the objective lens 10 are provided with laser light L4, L5 and laser light L6.
And a laser head 21, a laser head 22, a laser head 29, a photodetector 23, a photodetector 24 and a photodetector 30 It is arranged on the image plane of the sample 11 by the optical system. The dichroic mirror 27 has a laser beam L4
And the laser light L5, L6 and the fluorescent light F4.
The dichroic mirror 32 is a wavelength separator that reflects the laser light L5 and the fluorescent light F5 and transmits the laser light L6 and the fluorescent light F6. Dichroic mirror 27 and dichroic mirror 3
2, the optical path of the light source from the dichroic mirror 25, the dichroic mirror 26, and the optical path of the objective lens optical system from the dichroic mirror 31 are connected. Next, the spectral characteristics of the dichroic mirror 25, dichroic mirror 26, dichroic mirror 31, dichroic mirror 27 and dichroic mirror 32 will be described with reference to FIG. The dichroic mirror 25, the dichroic mirror 26 and the dichroic mirror 31 have wavelengths of 488 nm and 568 nm, respectively.
And the rising edge of the spectral transmittance curve is set on the nearest long wavelength side of 647 nm. Dichroic mirror 27
The rise of the spectral transmittance curve of the dichroic mirror 32 is set at around 560 nm and around 640 nm, respectively. The dichroic mirror 27 and the dichroic mirror 32 thus have extremely simple spectral characteristics and can be manufactured at low cost. Next, the operation will be described. The laser light L4 emitted from the laser head 21 is reflected by the dichroic mirror 25, then reflected by the dichroic mirror 27, introduced into the objective lens optical system, and the laser light L5 emitted from the laser head 22 is reflected by the dichroic mirror 26.
Then, the laser light L6 transmitted through the dichroic mirror 27 and introduced into the objective lens optical system after being reflected by the dichroic mirror 27 is reflected by the dichroic mirror 31 and then transmitted through the dichroic mirror 32 and introduced into the objective lens optical system. Is done. The laser beam L4, the laser beam L5, and the laser beam L6 are condensed by the objective lens 10 via the scanning unit 7, the relay lenses 8, 9, and irradiate the sample 11. Fluorescent light F4, fluorescent light F5, and fluorescent light F6 are emitted from the specimen 11 and condensed by the objective lens 10.
It moves backward through the relay lenses 8 and 9 and the scanning unit 7. The fluorescent light F4 is reflected by the dichroic mirror 27, passes through the dichroic mirror 25, and
Incident on. The fluorescent light F5 passes through the dichroic mirror 27, is reflected by the dichroic mirror 32, passes through the dichroic mirror 26, and enters the photodetector 24.
The fluorescent light F6 passes through the dichroic mirror 27, the dichroic mirror 32, and the dichroic mirror 31, and enters the photodetector 30. Then, a fluorescence image is obtained. In both embodiments, even when the sample is excited by a plurality of laser beams from a plurality of light sources, it is not necessary to use a dichroic mirror having a plurality of reflection bands. Therefore, the loss of the excitation light and the fluorescence by the dichroic mirror is reduced, and the acquisition rate of the fluorescence is particularly improved, so that a bright fluorescence image can be easily acquired. The light source is not limited to a laser, but may be a continuous light source, and excitation with an arbitrary wavelength can be performed efficiently. In both embodiments, since a light detector and a wavelength separator are provided corresponding to each light source, it is easy to exchange light sources integrally with the light detector and the wavelength separator, The range of choice of substances is expanded. The excitation light of the first wavelength from the first light source is separated by the first light separating means, and the excitation light of the second wavelength from the second light source is separated by the second light source. After being separated by the light separating means, the sample is guided to the same optical path by the third light separating means to irradiate the sample. The fluorescence of the second wavelength and the fluorescence of the fourth wavelength from the specimen are separated by the third light separating means. The fluorescence of the second wavelength is separated by the first light separating means and detected by the first light detecting means, and the fluorescence of the fourth wavelength is
Since the light is separated by the second light separating means and detected by the second light detecting means, the excitation light or the fluorescent light can be separated by an inexpensive wavelength separator with a small loss of light and can be separated. A fluorescent image can be easily obtained.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing an optical arrangement according to an embodiment of the present invention. FIG. 2 is a diagram showing a spectral intensity distribution of excitation light and fluorescence according to one embodiment of the present invention. FIG. 3 is a diagram illustrating spectral characteristics of a dichroic mirror according to one embodiment of the present invention. FIG. 4 is a diagram showing an optical arrangement according to a second embodiment of the present invention. FIG. 5 is a diagram illustrating spectral characteristics of a dichroic mirror according to a second example of the present invention. FIG. 6 is a diagram showing an optical arrangement of a conventional laser scanning fluorescence microscope. FIG. 7 is a diagram showing spectral characteristics of a dichroic mirror according to a conventional laser scanning fluorescence microscope. [Explanation of reference numerals] 1, 2, 3, 21, 22, 29,..., Laser heads 4, 5, 6, 15, 16, 25, 26, 27, 31,.
.. dichroic mirror 10 ... objective lens 11 ... specimens 12, 13, 14, 23, 24, 30 ... photodetectors L1, L2, L3, L4, L5, L6 ... Laser light F1, F2, F, F4, F5, F6 ... fluorescence

Claims (1)

(57) Claims 1. An excitation light having a first wavelength is generated, and a sample is irradiated to generate fluorescence having a second wavelength longer than the first wavelength. And a second light source that generates excitation light of a third wavelength longer than the second wavelength and irradiates the sample to generate fluorescence of a fourth wavelength longer than the third wavelength. A light source; first light separating means having spectral characteristics for separating light at a wavelength set between the first wavelength and the second wavelength; A second light separating unit having a spectral characteristic of separating light at a wavelength set between the second wavelength and the third wavelength; and a second light separating unit having a wavelength set between the second wavelength and the third wavelength. Having a spectral characteristic of separating light, and having the excitation light from the first light source and the excitation light from the second light source be the same light to the sample; And a third light separating means for separating light from the sample according to the spectral characteristics thereof, and one of the light from the sample, separated by the third light separating means, to the first light separating means. First light detecting means for detecting the other of the light from the sample, separated by the third light separating means, via the second light separating means. A fluorescence microscope comprising: