JP3952499B2 - Confocal scanner microscope - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to confocal scanner microscopes, and more particularly to improving image quality and sensitivity.
[0002]
[Prior art]
Conventionally, confocal scanner microscopes are well known (for example, see Patent Document 1). FIG. 4 is a principle configuration diagram of the main part of such a confocal scanner microscope. In FIG. 4, the laser beam emitted from the laser light source 1 first passes through an excitation filter (not shown) to remove the extra wavelength component of the light source and emit only the wavelength component used for excitation.
[0003]
The emitted light (laser light) is spread to an appropriate beam diameter and then enters the microlens array of the microlens array disk 2 of the scanner unit. The laser light is focused on the pinholes of the pinhole disk 3 arranged corresponding to each microlens.
[0004]
By this light collection, the amount of light that can pass through the pinhole disk 3 is greatly improved. At the same time, reflected light (noise light) on the disk surface other than the pinhole is reduced, and the S / N ratio is improved.
[0005]
In this case, between the microlens array disk 2 and the pinhole disk 3 is arranged a dichroic mirror 4, the excitation light is transmitted through the dichroic mirror 4.
[0006]
The laser beam 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 passes through the objective lens 5 and the pinhole of the pinhole disk 3 again, is reflected by the dichroic mirror 4, and is guided to the observation optical path system.
[0007]
The fluorescence emitted from the scanner unit to the observation optical path system enters the so-called HARP high-sensitivity imaging camera 8 using a HARP film after the reflection of reflected excitation light and stray light components mixed therein are removed by the barrier filter 7. A fluorescent image of the sample 6 is observed by the camera 8 (see, for example, Non-Patent Document 1 for the HARP film and Non-Patent Document 2 for the HARP camera).
Note that the microlens array disk 2 and the pinhole disk 3 are integrated, and the entire observation area can be scanned by rotation.
[0008]
[Patent Document 1]
Japanese Patent Laid-Open No. 5-60980 (page 3, FIG. 1)
[0009]
[Non-Patent Document 1]
"High-sensitivity HARP film for solid-state imaging devices", [online], [searched on August 27, 2002], Internet <URL: http://www.nhk.or.jp/strl/open99/ki-1/HARP .html>
[0010]
[Non-Patent Document 2]
"Ultrahigh-sensitivity HDTV Super-HARP Handheld Camera", [online],
[Search August 27, 2002] Internet <URL: http://www.nhk.or.jp/strl/open2002/en/tenji/id21/21.html>
[0011]
[Problems to be solved by the invention]
However, such a conventional apparatus has advantages such as improved imaging characteristics and reduced surface reflection from the pinhole disk surface for the confocal optical scanner part, but in the case of camera imaging, There was a problem like this.
(1) When there are few fluorescent molecules (one in the case of the minimum), the sensitivity is too low for real-time measurement, zoom-up, or three-dimensional measurement to observe.
(2) When the fluorescence from the sample is red, the sensitivity characteristics of HARP high-sensitivity imaging cameras (HARP is a method developed by Japan Broadcasting Corporation NHK) depend on the band gap of the semiconductor thin film. Therefore, it becomes 1/10 in the red band.
[0012]
The characteristics of a typical HARP imaging camera are shown in FIG. B / Gch uses amorphous selenium for the photosensitive film, and Rch adds tellurium, but the required sensitivity is 600 to 700 nm red and is extremely low, 1/4 to 1/1000 of green.
[0013]
An object of the present invention is to solve the above-described problems, and by using an image intensifier, the influence of sensitivity reduction due to the wavelength of a high-sensitivity imaging camera is eliminated, and a high-quality, high-sensitivity fluorescent image can be obtained. It is to realize a confocal scanner microscope that can be used.
Another object of the present invention is to provide a confocal scanner microscope that can easily identify the type of fluorescent substance in a sample by disposing a filter in the light detection section and changing the characteristics of the filter. There is.
[0014]
[Means for Solving the Problems]
In order to achieve such an object, in the present invention,
A light source unit that emits excitation light for exciting the sample, a scanner unit that scans the sample with a plurality of beams using the excitation light, and a sample that receives the fluorescence emitted from the sample with green and high sensitivity characteristics. In a confocal scanner microscope having an imaging camera that captures an image,
A HARP imaging camera;
An image intensifier that amplifies the fluorescence emitted from the sample and converts the input light to green and output;
A lens that is disposed between the image intensifier and the HARP imaging camera and collects output light of the image intensifier;
The apparatus has a means for receiving the output of the HARP imaging camera and displaying a fluorescent image of the sample on the screen.
[0015]
Since the fluorescence emitted from the sample is highly amplified by the image intensifier, it can be observed with high sensitivity. As in the prior art, when there are few fluorescent molecules, there is no problem that the sensitivity is too low to be observed.
Further, by interposing the image intensifier, since the converted all fluorescent inputs green incident on the image pickup camera of the HARP method also eliminates desensitization problems with red imaging camera HARP method There is an effect that all fluorescent images can be taken with high sensitivity.
[0016]
In this case, as in claim 2, a plurality of filters that individually transmit light of different wavelengths in the fluorescence emitted from the sample are provided, and light that has passed through the filter by switching each filter enters the image intensifier. Can be configured to.
When filters having different transmission wavelengths are used in this way, light of each wavelength of fluorescence can be easily taken out and imaged with a camera.
[0017]
Furthermore, the fluorescent substance of the sample can be identified by changing the transmission wavelength region by changing the characteristics of the filter.
[0018]
According to a fourth aspect of the present invention, there is provided a plurality of sets of detection systems including branching means for branching fluorescence emitted from the sample for each light of different wavelengths, the image intensifier, a lens, and a HARP imaging camera. And branching light from the branching unit can be separately guided to the detection system so that a plurality of branching lights can be observed simultaneously.
[0019]
According to a fifth aspect of the present invention, a plurality of barrier filters that narrow the transmission wavelength range and transmit light in the target wavelength range are provided, and output light of the branching means is given to the image intensifiers through the barrier filters. It can also be.
By using the barrier filter, light other than the target wavelength can be removed, and a high-quality image can be obtained.
[0020]
Further, as in claim 6, the means for displaying the fluorescent image of the sample on the screen can be configured to display the fluorescent image by performing color reconstruction.
[0021]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail with reference to the drawings. FIG. 1 is a conceptual block diagram showing an embodiment of a confocal scanner microscope according to the present invention. In FIG. 1, the same components as those in FIG. 4 are denoted by the same reference numerals. A difference from FIG. 4 is a light source unit 10, a light detection unit 20, an image collection device 30, and an image display device 40.
[0022]
The light source unit 10 includes a plurality of multi-wavelength light sources 11a, 11b, and 11c, filters 12a, 12b, and 12c having different light transmission bands, dichroic mirrors 13a, 13b, and 13c for mixing, and a switching mechanism 14. .
[0023]
R, G, and B wavelength component lights are extracted by passing the output lights of the multi-wavelength light sources 11a, 11b, and 11c through the filters 12a, 12b, and 12c, respectively. In the dichroic mirrors 13a, 13b, and 13c, the R, G, and B lights are combined and emitted as white light.
[0024]
The switching mechanism 14 controls the passage and blocking of the white light. The light that has passed through the switching mechanism 14 enters the mirror 16 via the optical fiber 15. The mirror 16 reflects this light and enters the lens 17. The lens 17 enters the microlens array disk 2 with incident light as parallel light.
[0025]
The light detection unit 20 includes a filter 21, an image intensifier 23, a lens 24, and the imaging camera 8.
The filter 21 is composed of a plurality of filters having different transmission wavelength characteristics, and each filter is configured to be alternatively selectable. As the selection mechanism, for example, a mechanism in which a plurality of filters are arranged on a disk and the filters are switched by rotation of the disk can be used.
[0026]
During the filter switching period, the combined light is blocked by the switching mechanism 14 of the light source unit 10, and the excitation light irradiation to the sample 6 is stopped. In addition, when it is not necessary to interrupt | block, you may make it the state of always passing, or you may remove the switching mechanism 14. FIG.
[0027]
The image intensifier 23 amplifies photons about 100 times. The amplification factor is substantially constant in the visible light range, and the output is a green fluorescent light emitter. Since the output is a green fluorescent emitter, all incident light is converted to green.
The output light of the image intensifier 23 is collected by the lens 24 and enters the imaging camera 8.
[0028]
The imaging camera 8 amplifies the captured image, converts it into an electrical signal, and outputs it. Note that the sensitivity characteristic of an imaging camera such as the HARP method depends on the band gap of the semiconductor thin film, and decreases to ¼ to 1/1000 in red. However, in the present invention, as described above, since the imaging camera captures only green light, the sensitivity is not affected by red sensitivity.
[0029]
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.
[0030]
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 obtained through the filters 12a, 12b, and 12c, respectively, and R, G, and B light is obtained, and is obtained by the dichroic mirrors 13a, 13b, and 13c. Synthesize. The synthesized white light passes through the switching mechanism 14 and enters the optical fiber 15.
[0031]
The light emitted from the optical fiber 15 is reflected by the mirror 16 and enters the lens 17. The light converted into parallel light by the 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.
[0032]
The operation from the time when the output light of the lens 17 is incident on the microlens array disk 2 until the fluorescence from the sample 6 is reflected by the dichroic mirror 4 and taken out is the same as in the conventional apparatus, and the description thereof is omitted here. .
[0033]
The fluorescence emitted from the sample 6 usually includes a plurality of wavelengths. Therefore, the filter 21 is switched to transmit the fluorescence of each wavelength individually. The fluorescence transmitted through the filter is amplified by the image intensifier 23, and the output light is collected by the lens 24 and enters the imaging camera 8. A fluorescent image of the sample 6 formed on the image receiving surface (not shown) of the imaging camera 8 is taken.
[0034]
The output light of the image intensifier 23 is always green regardless of the transmitted light of the filter. The imaging camera 8 captures the fluorescent image converted to green. In the image acquisition device 30, information on the type (or transmission wavelength) of the selected filter and the output image (green image) signal of the imaging camera 8 are stored in association with each other.
[0035]
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.
[0036]
In this way, the fluorescence image can be observed by changing the transmission wavelength of the filter in a time division manner. At this time, it is also possible to identify the type of fluorescent material by changing the characteristics of the filter.
[0037]
Furthermore, since the fluorescence is highly amplified by the image intensifier in the present invention, even if there are few fluorescent molecules, it can be displayed as an image and can be observed reliably. In addition, since all fluorescence is converted to green by the image intensifier, the imaging camera can capture all fluorescence with the same sensitivity without being affected by red sensitivity reduction as in the past. it can.
[0038]
Further, by using the intensifier 23 and the lens 24, it is possible to obtain a sensitivity of about 100 times while maintaining a higher image quality than before.
In addition, the sensitivity in the long wavelength (red) region below the band gap of the photoconductive film has no sensitivity drop on the long wavelength side of the imaging camera. Along with the 100 times increase in fire sensitivity, the present invention achieves a sensitivity increase of about 1000 times.
[0039]
FIG. 2 shows another embodiment of the present invention. In the embodiment shown in FIG. 1, fluorescence having different wavelengths is measured in a time-sharing manner by changing the wavelength of the filter, whereas in this embodiment, fluorescence having different wavelengths is simultaneously measured using a dichroic mirror. Yes.
[0040]
2 differs from FIG. 1 in the light detection unit 20a. The light detection unit 20a includes three sets of detection systems, that is, three sets of detection systems including a dichroic mirror, an image intensifier, a lens, and an imaging camera.
[0041]
The dichroic mirrors 22a, 22b, and 22c branch light of three wavelengths included in the fluorescence. In this embodiment, other than three branches are possible according to the number of wavelengths. In that case, a dichroic mirror, an image intensifier, a lens, and an imaging camera corresponding to the number of branches are required.
[0042]
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.
[0043]
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. In addition, each image intensifier is arrange | positioned so that the input surface may correspond to the said real image surface of each fluorescence.
[0044]
Each image intensifier 23a, 23b, 23c has the same characteristics as the image intensifier of FIG. The output light of each image intensifier is condensed by the lenses 24a, 24b, and 24c and enters the imaging cameras 8a, 8b, and 8c.
[0045]
The imaging cameras 8a, 8b, and 8c have the same sensitivity characteristics as the imaging camera 8 of FIG. 1, and respectively capture green light from the image intensifier. Therefore, all fluorescence can be imaged with the same sensitivity.
[0046]
Each of the three imaging cameras converts the captured image (green 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 the image data stored in the image collection device 30 and performs an appropriate process to display a fluorescent image on the screen. At this time, the image is reconstructed (color reconstructed) into a color associated with the image and displayed.
[0047]
In this way, it is possible to simultaneously measure fluorescent images of wavelengths branched by the dichroic mirror.
In the present embodiment, as in the case of FIG. 1, high image quality and high sensitivity can be easily realized.
[0048]
FIG. 3 shows still another embodiment of the present invention. The difference from FIG. 2 is that barrier filters 25a, 25b, and 25c are arranged in front of the image intensifiers 23a, 23b, and 23c, respectively. By narrowing the transmission wavelength region with this barrier filter, it is possible to remove the wavelengths other than the target wavelength and make only the target wavelength incident on the image intensifier, thereby improving the SN ratio of the image.
[0049]
The present invention is not limited to the above-described embodiments, and includes many changes and modifications without departing from the essence thereof.
[0050]
【The invention's effect】
As described above, the present invention has the following effects.
(1) The image intensifier and the lens that condenses the output light are arranged so that the HARP imaging camera captures all real images converted to green so that high image quality can be easily maintained. However, it is possible to obtain an image with a sensitivity about 100 times that of the conventional apparatus.
[0051]
(2) Further, since the long wavelength (red) region below the band gap of the photoconductive film is not photographed with the HARP imaging camera, the sensitivity drop on the long wavelength side with the HARP imaging camera as in the conventional apparatus. There is no. Equivalently, the sensitivity in the long wavelength region is about 1000 times. Incidentally, this is also an imaging camera other methods less sensitive to red as well HARP method, the same effect can be obtained, it is effective.
[0052]
(3) In addition, the fluorescent substance can be easily identified by changing the transmission wavelength characteristic of the filter in the embodiment of FIG.
(4) By arranging a barrier filter in front of the image intensifier to narrow the transmission wavelength region, it is possible to easily increase the SN ratio of the image.
(5) Also, by obtaining a plurality of types of screens with different filters or screens branched by dichroic mirrors, it is possible to easily colorize the captured image.
[Brief description of the drawings]
FIG. 1 is a configuration diagram showing an embodiment of a confocal scanner microscope according to the present invention.
FIG. 2 is a block diagram showing another embodiment of the present invention.
FIG. 3 is a block diagram showing still another embodiment of the present invention.
FIG. 4 is a configuration diagram of a main part of a conventional confocal scanner microscope.
FIG. 5 is a diagram illustrating an example of characteristics of a HARP imaging camera.
[Explanation of symbols]
2 Microlens array disk 3 Pinhole disk 4 Dichroic mirror 5 Objective lens 6 Sample 8, 8a, 8b, 8c Imaging camera 10 Light source 11a, 11b, 11c Multi-wavelength light source 12a, 12b, 12c Filter 13a, 13b, 13c Dichroic mirror 14 Switching mechanism 15 Optical fiber 16 Mirror 17 Lens 20, 20a, 20b Photodetector 21 Filter 22a, 22b, 22c Dichroic mirror 23, 23a, 23b, 23c Image intensifier 24, 24a, 24b, 24c Lens 25a, 25b, 25c Barrier filter 30 Image collection device 40 Image display device

Claims (6)

A light source unit that emits excitation light that excites the sample, a scanner unit that scans the sample with a plurality of beams using the excitation light, and a fluorescent light emitted from the sample that has high sensitivity and is green. In a confocal scanner microscope having an imaging camera that captures an image,
A HARP imaging camera;
An image intensifier that amplifies the fluorescence emitted from the sample and converts the input light to green and output;
A lens that is disposed between the image intensifier and the HARP imaging camera and collects output light of the image intensifier;
A confocal scanner microscope comprising means for receiving the output of the HARP imaging camera and displaying a fluorescent image of a sample on a screen.   A plurality of filters that individually transmit light of different wavelengths in the fluorescence emitted from the sample are provided, and each filter is switched so that light transmitted through the filter is incident on the image intensifier. The confocal scanner microscope according to claim 1.   The confocal scanner microscope according to claim 2, wherein the fluorescent substance of the sample can be identified by changing the transmission wavelength region by changing the characteristics of the filter. Branching means for branching fluorescence emitted from the sample for each light of different wavelengths;
A plurality of detection systems comprising the image intensifier, lens, and HARP imaging camera;
The confocal scanner microscope according to claim 1, wherein the branched light from the branching means is separately guided to the detection system so that a plurality of branched lights can be observed simultaneously.   5. The confocal system according to claim 4, further comprising a plurality of barrier filters that narrow a transmission wavelength range and transmit light in a target wavelength range, and output light from a branching unit is supplied to each image intensifier through each barrier filter. Scanner microscope.   6. The confocal scanner microscope according to claim 2, wherein the means for displaying the fluorescent image of the sample on the screen is configured to display a fluorescent image by performing color reconstruction.

Images