JP5083618B2 - Confocal scanner microscope - Google Patents

Description

  The present invention relates to a confocal scanner microscope, and more particularly to a confocal scanner microscope capable of high-speed imaging even when all or part of a multi-wavelength light source includes a light source having a weak output.

In recent years, confocal scanner microscopes have attracted attention. The confocal scanner microscope includes a light source unit that excites a sample, a light detection unit that detects fluorescence emitted from the sample, and the following Patent Document 1 discloses an invention of a confocal scanner microscope that performs imaging using a multi-wavelength light source. Have been described.

JP 2004-212434 A

Since the present invention is an improvement of the light source portion of the invention described in FIG. 1 of Patent Document 1, first, the invention described in FIG. 1 of Patent Document 1 will be described.

FIG. 8 is a block diagram showing an embodiment of a conventional confocal scanner microscope. The light source unit 10 includes a plurality of multi-wavelength light sources 11a, 11b, and 11c, switching means 14 that controls passage and blocking of light from the light sources, and filters 12a, 12b, and 12c having different light transmission bands.

  The light that has passed through the switching means 14 passes through the filters 12a, 12b, and 12c, and enters the optical fibers 15a, 15b, and 15c separated from the optical fiber bundle 15 into a plurality of optical fibers corresponding to the number of light sources. The light exits from the other end of 15 and enters the mirror 16. The mirror 16 reflects this light and enters the collimator lens 17. The collimator lens 17 enters the microlens array disk 2 with the incident light as parallel light.

The incident laser light is reduced in the reflected light (noise light) on the surface of the pinhole disk disposed corresponding to each microlens, and the S / N ratio is improved. A dichroic mirror 4 is disposed between the microlens array disk 2 and the pinhole disk 3, and excitation light passes through the dichroic mirror 4.

The laser light exiting the pinhole of the pinhole disk 3 excites the sample 6 after passing through the objective lens 5. The fluorescence emitted from the sample 6 again passes through the objective lens 5 and the pinhole 3 of the pinhole disk 3, is reflected by the dichroic mirror 4, and is guided to the light detection unit 20.

  The light detection unit 20 includes dichroic mirrors 22a, 22b, and 22c, barrier filters 25a, 25b, and 25c, lenses 24a, 24b, and 24c, and imaging cameras 8a, 8b, and 8c. The barrier filter 25 is composed of a plurality of filters having different transmission wavelength characteristics. The output light that has passed through the barrier filter 25 is collected by the lens 24 and enters the imaging camera 8. The imaging camera 8 amplifies the captured image, converts it into an electrical signal, and outputs it.

  The image collecting device 30 converts the electrical signal output from the imaging camera 8 into image data and stores it. The image display device 40 is usually a computer, reads an image stored in the image collection device 30, performs appropriate processing, and displays it on a display screen.

  The operation in such a configuration will be described next. In the light source unit 10, light emitted from the three multi-wavelength light sources 11a, 11b, and 11c is incident on one end of optical fibers 15a, 15b, and 15c separated into a plurality of parts through the switching means 14 and the filters 12a, 12b, and 12c, respectively. To do.

  The light emitted from the optical fiber bundle 15 is reflected by the mirror 16 and enters the collimator lens 17. The light that has been collimated by the collimator lens 17 enters the microlens array disk 2. If the output light of the optical fiber 15 can be directly incident on the lens 17, the mirror 16 is not necessary.

  The fluorescence emitted from the sample 6 usually includes a plurality of wavelengths. For example, the first dichroic mirror 22a transmits light of wavelength λ1, and reflects light of other wavelengths. Next, the second dichroic mirror 22b receives light reflected by the first dichroic mirror 22a, reflects light of wavelength λ2, and transmits light of other wavelengths. The third dichroic mirror 22c receives the light transmitted through the second dichroic mirror 22b and reflects the light having the wavelength λ3.

  In this way, the light is branched by the three dichroic mirrors 22a, 22b, and 22c, and the three wavelengths of fluorescence are individually output. The output from the dichroic mirror 22 is input to the barrier filters 25a, 25b, and 25c. By narrowing the transmission wavelength region with this barrier filter 25, it is possible to remove the wavelength other than the target wavelength and make only the target wavelength incident on the imaging camera 8, and to improve the SN ratio of the image.

  The respective output lights that have passed through the barrier filter are condensed by the lenses 24a, 24b, and 24c, and enter the imaging cameras 8a, 8b, and 8c. Each of the three imaging cameras converts the captured image into an electrical signal and outputs it. The image acquisition device 30 converts this electrical signal into digital image data, and stores it in association with the fluorescence color branched by the dichroic mirror.

  The image display device 40 reads out image data and filter information stored in the image collection device 30, reconstructs the original fluorescence color (color reconstruction), and displays the fluorescence image on the screen. It is of course possible to display a fluorescent image without color reconstruction. In this way, it is possible to simultaneously measure fluorescent images of wavelengths branched by the dichroic mirror.

  Next, the shift amount of the light output from the optical fiber bundle will be described. In FIG. 9, there is a deviation of 125 μm between the cores when the three are bundled. A smaller shift is advantageous for a clear image display. However, it is technically and economically difficult to make the clad thinner and bring the cores closer together.

  FIG. 10 shows a conventional scanner unit composed of a collimator lens 17, a microlens array disk 2, a pinhole disk 3, and an objective lens, which emits light from an optical fiber bundle when the core interval has a deviation of 125 μm. It is an image figure which shows the result of having calculated | required the change of the deviation | shift amount of an optical axis by calculation.

  The light collimated by the collimator lens 17 enters the microdisk lens 2. The deviation becomes 33.5 μm at this incident time. Here, when the focused light passes through the pinhole disk 3, it decreases to 7 μm, and when the sample is irradiated through the objective lens (40 times) 5, there is a shift of 0.26 μm. These values are calculated from the data of the lens currently used.

  FIG. 11 shows the cross-sectional intensity distribution of the focused spot (a) on the surface of the pinhole disk 3 and the light (b) immediately after passing through the pinhole disk 3, and shows the diameter of the pinhole currently used. In this case, the transmission (attenuation) state of light having a deviation of 7 μm is shown (the point G shown in FIG. B shows the state of the waveform cut by the deviation). According to the calculations by the inventors, the transmittance when there is no deviation is 86.9%, 7 μm, and the transmittance when there is a deviation is 86.3%. Even if the optical axis deviation is 7 μm, there is almost no influence. I understand that there is no.

As described above, the conventional confocal scanner microscope can perform imaging using a multi-wavelength light source.

However, when a light source with low output light power or a light source with low optical coupling with an optical fiber is used, the light power emitted from the optical fiber bundle becomes weak, so it is necessary to lengthen the exposure time of the imaging camera. This is not suitable for high-speed imaging.

This problem is that when a light source with low output light power or a light source with low optical coupling with an optical fiber (for example, the light sources 11c and 11d in FIG. 12) is used, a plurality of the same light sources 11c and 11d are prepared as shown in FIG. Can respond. However, when a plurality of the same light sources are used, the multicolor excitation is limited. Further, if the number of optical fibers to be bundled is increased in order to increase the types of light sources, the deviation between the optical fiber cores increases, which is disadvantageous for obtaining a clear image.

The present invention has been made in view of these problems, and an object of the present invention is to provide a confocal scanner microscope capable of high-speed imaging even when all or a part of a multi-wavelength light source includes a light source having a weak output.

In order to achieve such a problem, the invention described in claim 1
A light source unit that excites a sample with a plurality of excitation lights having different wavelengths, a scanner unit that scans the sample using the excitation light, an imaging camera that receives fluorescence emitted from the excited sample and captures an image of the sample, and In a confocal scanner microscope with
The light source unit is
A first light source and a second light source that output excitation light having a first wavelength of the plurality of wavelengths;
A light source group that outputs excitation light having a wavelength different from the first wavelength among the plurality of wavelengths;
A first wavelength-selective optical fiber coupler that synthesizes the excitation light of the first wavelength output from the first light source and the excitation light output from one light source included in the light source group;
A second wavelength-selective optical fiber coupler that synthesizes the excitation light of the first wavelength output from the second light source and the excitation light output from another light source included in the light source group; ,
A first optical fiber for transmitting the synthesized pumping light via the first wavelength selective optical fiber coupler;
A second optical fiber for transmitting the synthesized pumping light via the second wavelength selective optical fiber coupler;
With
The first optical fiber and the second optical fiber constitute a common optical fiber bundle.

The invention according to claim 2
A light source unit that excites a sample with a plurality of excitation lights having different wavelengths, a scanner unit that scans the sample using the excitation light, an imaging camera that receives fluorescence emitted from the excited sample and captures an image of the sample, and In a confocal scanner microscope with
The light source unit is
A first light source and a second light source that output excitation light having a first wavelength of the plurality of wavelengths;
A third light source that outputs excitation light having a wavelength different from the first wavelength among the plurality of wavelengths;
An optical fiber coupler that combines the excitation light of the first wavelength output from the first light source and the excitation light of the first wavelength output from the second light source;
A first optical fiber for transmitting the synthesized pumping light via the first optical fiber coupler;
A second optical fiber that transmits excitation light output from the third light source;
With
The first optical fiber and the second optical fiber constitute a common optical fiber bundle.

The present invention has the following effects. Since the wavelength selective optical fiber coupler is used to increase the intensity of the excitation light, high-speed imaging is possible.

Hereinafter, a confocal scanner microscope according to the present invention will be described with reference to the drawings. FIG. 1 is a block diagram of a first embodiment of the present invention. The light sources 11c, 11d, 11e, and 11f are light sources with low output light power and light sources with low optical coupling with optical fibers. The light sources 11c and 11e and 11d and 11f are light sources that output the same wavelength.

When the wavelength-selective optical fiber couplers 50a and 50b enter the light source 11c having the wavelength λ3 from the port A and the light source 11d having the wavelength λ4 from the port B, the combined light of the wavelengths λ1 and λ2 from the port C (λ1 + λ2) Is output.

At this time, the transmittance from port A to port C and from port B to port C are both 90% or more. An optical fiber coupler having such characteristics is generally called a wavelength selective optical fiber coupler. A wavelength-selective optical fiber coupler is generally manufactured by bundling two optical fibers, heating, stretching, and fusing them. The mode coupling between the two optical fibers is wavelength-dependent. Use to achieve wavelength selectivity.

A specific example of FIG. 1 is shown below. For example, when the light source 11c and the light source 11e are light sources having the same optical power, if the switching means 14c and 14e are made through, the transmittance of the wavelength selective optical fiber coupler 50 is generally 90% or more, so one light source is used. Compared with the case where it is used, the optical power slightly less than twice is emitted from the optical fiber bundle 15. In addition, when the switching means 14 c and 14 d are made through, the light combined by the wavelength selective optical fiber coupler 50 is emitted from the optical fiber bundle 15. In addition, since the operation | movement after radiate | emitting from the optical fiber bundle 15 is the same as that of FIG. 8, description is abbreviate | omitted.

In FIG. 1, the wavelength selective optical fiber coupler is connected to the port 3 and the port 4 of the optical fiber bundle, but may be used at any port of the optical fiber bundle. The wavelength selective optical fiber coupler 50 and the port of the optical fiber bundle 15 may be connected by any joining method such as optical connector connection or fusion connection. Furthermore, although two sets of wavelength selective optical fiber couplers 50 are used in FIG. 1, they may be increased as necessary.

Further, as shown in FIG. 1, the unnecessary port D may be terminated so as not to be reflected by the end face of the optical fiber by, for example, oblique cutting. As another method, a light detector may be connected to port D to monitor the light output power of the light source.

  Next, another embodiment of the present invention will be described with reference to FIG. FIG. 2 shows a configuration using an optical fiber coupler used for optical coupling or branching instead of the wavelength selective optical fiber coupler of FIG. Due to the characteristics of this optical fiber coupler, a light source having the same wavelength can be used. Such an optical fiber coupler is manufactured by bundling two optical fibers together like a wavelength-selective optical fiber, heating, melting, stretching, and fusing.

The difference from the wavelength selective optical fiber is that the branching ratio is adjusted by the close distance (coupling length) between the fiber cores. Specifically, when the transmittance from port A to port C is 90%, the transmittance from port B to port C is 10% due to reciprocity.

In the invention of FIG. 2 as well, the light intensity of the light source with low output light power can be increased, so that the exposure time of the imaging camera can be shortened, and as a result, high-speed imaging is advantageous.

By the way, although the optical fiber bundle described in patent document 1 is a 3 configuration, in this invention, it is a 4 configuration. Therefore, the distance between the cores of the present invention is longer due to the influence of the clad diameter, and the optical axis is deviated. In general, the smaller the deviation of the optical axis, the better. Therefore, in the following description, the influence of the widening of the deviation of the optical axis is examined. Since the wavelength selective optical fiber coupler is composed of an optical fiber, the optical fiber bundle itself does not cause an optical axis shift.

FIG. 3A is a cross-sectional view of an optical fiber bundle having three configurations, and FIG. 3B is a cross-sectional view of an optical fiber bundle having four configurations. When the number of optical fibers is four, the maximum inter-core distance becomes 177 μm because the route is three times the route of 125 μm.

  FIG. 4 is a table in which the effects of having four optical fibers having three configurations are verified. According to the calculations by the inventors, it can be seen that the optical axis is shifted by 9.7 μm at the incident surface of the pinhole disk 3 when the distance between the cores is 177 μm. This is illustrated in FIG. FIG. 5A illustrates a conventional optical axis shift, and is provided for comparison with FIG. 5B. On the other hand, FIG. 5B is a diagram for explaining the deviation of the optical axis of the present invention.

  In FIG. 5, the transmittance of the pinhole disk 3 is 1. If there is no deviation, it is 86.9%. In the case of the conventional three-line configuration (that is, when shifted by 7 μm), it is 86.3%, and in the case of the four-line structure (that is, when shifted by 9.7 μm) as shown in FIG. Therefore, it can be seen that the four configuration has almost no influence compared to the three configuration.

  FIG. 6 is a structural diagram of the second embodiment of the present invention. The difference from FIG. 1 is that a polarization maintaining optical fiber is used for the optical fiber bundle. The polarization axes (slow axes) of the port 3 and the port 4 of the optical fiber bundle 15 are orthogonally arranged as shown in FIG. 7 showing the Z view of FIG. Note that the polarization axis of the light source 11 and the slow axis of the optical fiber bundle 15 coincide.

  According to the second embodiment, for example, when the light sources 11c and 11e, 11d and 11f are laser light sources having high coherence and the same wavelength, respectively, a collimator lens (not shown) arranged at the rear stage of the optical fiber bundle (FIG. 8). The interference noise generated spatially can be suppressed as much as possible.

As described above, according to the present invention, the intensity of pumping light can be increased by using the wavelength selective optical fiber coupler 50, so that high-speed imaging is possible.

It is a block diagram of the light source part of this invention. It is drawing which supplements description of the light source part of this invention. (A) is a cross-sectional view of a three-configuration optical fiber bundle, and (B) is a cross-sectional view of a four-configuration optical fiber bundle. It is the table | surface which verified the demerit by having increased the distance between cores. (A) explains the conventional optical axis deviation, and (B) is a drawing explaining the optical axis deviation of the present invention. It is a structural diagram of the 2nd example of the present invention. FIG. 7 is a Z view of FIG. 6 for explaining a state in which they are arranged orthogonally. It is a block diagram of the conventional confocal scanner microscope. It is drawing explaining the shift | offset | difference of the optical axis of the conventional three single mode fiber. It is an image figure which shows a mode that the space | interval of a core narrows with the combination of a lens. It is a figure which shows the cross-sectional intensity distribution of the light on a pinhole disk surface and immediately after pinhole transmission. This is an example of a multi-wavelength light source including light sources 11c and 11d having low output light power. This is an example in which a plurality of light sources 11c and 11d in FIG.

Explanation of symbols

2 Microlens array disk 3 Pinhole disk 4 Dichroic mirror 5 Objective lens 6 Sample 8 Video camera 10 Light source 11 Multi-wavelength light source 12 Filter 14 Switching means 15 Optical fiber bundle 16 Mirror 17 Lens 20 Photodetector 22 Dichroic mirror 24 Lens 25 Barrier filter 30 Image collection device 40 Image display device

Claims (2)

A light source unit that excites a sample with a plurality of excitation lights having different wavelengths, a scanner unit that scans the sample using the excitation light, an imaging camera that receives fluorescence emitted from the excited sample and captures an image of the sample, and In a confocal scanner microscope with
The light source unit is
A first light source and a second light source that output excitation light having a first wavelength of the plurality of wavelengths;
A light source group that outputs excitation light having a wavelength different from the first wavelength among the plurality of wavelengths;
A first wavelength-selective optical fiber coupler that synthesizes the excitation light of the first wavelength output from the first light source and the excitation light output from one light source included in the light source group;
A second wavelength-selective optical fiber coupler that synthesizes the excitation light of the first wavelength output from the second light source and the excitation light output from another light source included in the light source group; ,
A first optical fiber for transmitting the synthesized pumping light via the first wavelength selective optical fiber coupler;
A second optical fiber for transmitting the synthesized pumping light via the second wavelength selective optical fiber coupler;
With
The confocal scanner microscope, wherein the first optical fiber and the second optical fiber constitute a common optical fiber bundle . A light source unit that excites a sample with a plurality of excitation lights having different wavelengths, a scanner unit that scans the sample using the excitation light, an imaging camera that receives fluorescence emitted from the excited sample and captures an image of the sample, and In a confocal scanner microscope with
The light source unit is
A first light source and a second light source that output excitation light having a first wavelength of the plurality of wavelengths;
A third light source that outputs excitation light having a wavelength different from the first wavelength among the plurality of wavelengths;
An optical fiber coupler that combines the excitation light of the first wavelength output from the first light source and the excitation light of the first wavelength output from the second light source;
A first optical fiber that transmits the synthesized pumping light via the optical fiber coupler;
A second optical fiber that transmits excitation light output from the third light source;
With
The confocal scanner microscope, wherein the first optical fiber and the second optical fiber constitute a common optical fiber bundle .

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