JPH1152252A - Fluorescent microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a bright multiple fluorescent image without the positional deviation of a sample being multiply dyed by changing over dichroic mirrors. SOLUTION: In a fluorescent microscope provided with a light source 1 emitting an excited laser beam, plural dichroic mirrors 13 reflecting the excited laser beam, transmitting a fluorescent beam through and changeably provided in an optical path, an XY scanner 14 for scanning the excited laser beam on a sample in a two-dimensional manner, an objective lens 18 arranged between the XY scanner 14 and the sample and a monitor for observing the fluorescent light beam, a controller 50 for inputting the information as to the kinds of the dichroic mirrors arranged in the optical path at the time of accquiring an image, a memory 33 for storing the information on an image deviation in the dichroic mirrors at the time of accquiring the image and a computer 30 for reading out the information on the image deviation stored in the memory 33 based on the information from the controller 50 and controlling the XY scanner 14 so that the images obtained by the different kinds of the dichronic mirrors are superimposed, are provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fluorescence microscope.

[0002]

2. Description of the Related Art A conventional fluorescence microscope is composed of a light source that emits excitation light, a plurality of dichroic mirrors that reflect excitation light, transmit fluorescence, and are switchably provided in an optical path, a dichroic mirror, and a sample. And an observation device for observing the fluorescence that has passed through the dichroic mirror.

Conventionally, in order to observe a sample which has been subjected to multiple fluorescent staining, an image is obtained by switching a dichroic mirror for each fluorescent dye used, and a plurality of obtained images are superimposed to obtain a multiple fluorescent image. A method is used in which a so-called multi-band filter such as a dual band filter capable of excitation at two different wavelengths or a triple band filter capable of excitation at three wavelengths is used to acquire multiple fluorescent images at a time. ing.

[0004]

In the above-described method of switching dichroic mirrors for each fluorescent dye, the mirror reflection angle changes little by little due to mechanical accuracy such as processing accuracy and mounting accuracy of each dichroic mirror. There is also a problem that a displacement occurs between the acquired images, and a plurality of images cannot be accurately superimposed.

As a method for solving this problem, it is conceivable that each acquired image is taken into a computer, and an operator corrects the amount of displacement of each image by visual measurement using image processing software.

[0006] However, this work is very troublesome because the shape of the sample shown in each image is not exactly the same as it is very time-consuming.

[0007] In the method of obtaining a multiple fluorescence image at a time by using the above-mentioned multi-band filter, a transmission wavelength range and a reflection wavelength range appear alternately, so that the wavelength range that can be transmitted is limited.

FIG. 5 is a diagram showing an example of transmission / reflection characteristics of a triple band filter, FIG. 6 is a diagram showing absorption and fluorescence intensity characteristics of a fluorescent dye FITC, and FIG.
FIG. 8 shows the absorbance and fluorescence intensity characteristics of the fluorescent dye Cy5.

For example, using the triple band filter shown in FIG. 5, the fluorescent dyes FITC, Cy3.5, C
When observing a sample triple-stained with y5, the characteristics of the triple band filter and the fluorescence emitted from the sample do not completely match, so that the fluorescence detected by the detector decreases.

Since the fluorescence emitted from the sample is weak, there is a problem that the image of the sample observed by the observation device becomes dark due to the decrease in the fluorescence.

This problem also occurs with a dual band filter.

[0012] Further, it is difficult to manufacture a dual band filter or a triple band filter by specifying a combination of transmission wavelength and reflection wavelength in detail, so that it is difficult to obtain an optimum filter according to a fluorescent dye. (For example, when the wavelengths of the reagents used are very close, a corresponding filter cannot be manufactured).

The present invention has been made in view of such circumstances, and a problem thereof is to provide a fluorescent microscope capable of obtaining a bright multiple fluorescence image without displacement of a multiple-stained sample by switching dichroic mirrors. It is to provide.

[0014]

According to a first aspect of the present invention, there is provided a fluorescence microscope comprising a light source for emitting excitation light, a stage on which a sample is mounted, and a switchable optical path. Obtaining an image by using a fluorescence microscope having a plurality of light separation units that reflect excitation light from the light source and transmit fluorescence from the sample, and observation units that obtain images of the fluorescence transmitted through the light separation unit. Input means for inputting information on the type of the light separating means arranged in the optical path, storage means for storing image shift information at the time of image acquisition in each of the light separating means, and information from the input means. Based on the information, the image shift information stored in the storage means is read, and the observation means or the scanning means is arranged so that images obtained by different types of light separation means are superimposed. And having a control means for controlling the over-di.

Input to the control means by the input means,
Information such as an error corresponding to the type of the light separating means is stored, and at the time of acquiring an image, the observation means or the stage is controlled based on this information to correct a shift of the acquired image.

According to a second aspect of the present invention, there is provided a fluorescence microscope, comprising: a light source which emits an excitation laser beam; and a plurality of light separation means which reflect the excitation laser beam, transmit fluorescence, and are switchably provided in an optical path. Means, scanning means for two-dimensionally scanning the sample with the excitation laser light, an objective lens arranged between the scanning means and the sample, and observation means for obtaining an image of fluorescence transmitted through the light separating means. In a fluorescence microscope comprising, input means for inputting information on the type of the light separating means arranged in the optical path at the time of image acquisition,
A storage unit for storing image shift information at the time of image acquisition in each of the light separating units, and reading out the image shift information stored in the storage unit based on information from the input unit; And control means for controlling the scanning means so that the images obtained by the scanning are superimposed.

Input to the control means by the input means,
Information such as an error corresponding to the type of the light separating means is stored, and at the time of acquiring an image, the scanning means is controlled based on this information to correct a deviation of the acquired image.

According to a third aspect of the present invention, there is provided the fluorescence microscope according to the first or second aspect, wherein the input means includes a detection means for identifying a type of the light separation means arranged in the optical path. It is characterized by.

The type of the light separating means is identified by the detecting means for identifying the type of the light separating means arranged in the optical path.

According to a fourth aspect of the present invention, there is provided the fluorescence microscope according to the second aspect, wherein the scanning means includes a galvano-type scanning mirror, and the control means controls the shift information stored in the storage means. The offset voltage for controlling the reflection angle of the galvano-scanning mirror is adjusted based on the voltage.

Since the offset voltage for controlling the reflection angle of the galvano scanning mirror is adjusted based on the displacement information stored in the storage means, the displacement can be accurately corrected by analog controlling the galvano scanning mirror.

According to a fifth aspect of the present invention, in the fluorescence microscope according to the first aspect, the observation means selects an image acquisition area from an area where the light from the light source irradiates the sample, The control unit controls a timing at which the observation unit starts acquiring an image based on the shift information stored in the storage unit.

An image acquisition area to be actually acquired as an image is selected from an area where the light from the light source irradiates the sample, and the image acquisition area by the observation means is shifted based on the shift information stored in the storage means.

According to a sixth aspect of the present invention, in the fluorescence microscope according to the first aspect, the control means controls the position of the stage based on the displacement information stored in the storage means. It is characterized by.

Based on the shift information stored in the storage means,
The stage is offset vertically and horizontally and the images are superimposed.

According to a seventh aspect of the present invention, there is provided the fluorescent microscope according to any one of the first to sixth aspects, further comprising a graphic pattern arranged on a primary image plane of the objective lens. Means for obtaining an image of the figure pattern for each of the light separating means, calculating a shift amount of another image based on one of the obtained images, and storing the shift amount in the storage means as shift information. Features.

The shift amount of each light separating means is calculated from the shift amount of the graphic pattern and stored, and when the image is obtained, the observation means, the stage or the scanning means is controlled based on the stored shift amount.

[0028]

Embodiments of the present invention will be described below with reference to the accompanying drawings.

FIG. 1 is a block diagram of a confocal laser scanning microscope which is an embodiment of the fluorescence microscope of the present invention.

This confocal laser scanning microscope comprises a laser light source 1 and a confocal laser scanning microscope main body (microscope main body) 1.
0 and a computer 30.

The microscope body 10 includes an excitation filter 11 and
A beam expander 12, a dichroic mirror (light separating means) 13, an XY scanner (scanning means) 14,
It includes a scanner driver 15, a condenser lens 16, a graphic pattern target 17, an objective lens 18, a barrier filter 19, a condenser lens 20, a pinhole 21, and a detector 22.

The excitation filter 11 has a characteristic of passing only laser light of a predetermined wavelength emitted from the laser light source 1.

The beam expander 12 has lenses 12a,
12b, and the laser beam is sized to fill the pupil plane of the objective lens.

The dichroic mirror 13 reflects the laser light and transmits the fluorescence emitted from the excited sample. The dichroic mirror 13 includes a plurality of dichroic mirrors A, B, C,... Having different characteristics, each of which is provided so as to be insertable into and removable from the optical path.

The XY scanner 14 is composed of galvano-type scanning mirrors 14A and 14B.
Perform a dimensional scan.

The scanner driver 15 comprises galvanometer drivers 15A and 15B for driving the galvanometer scanning mirrors 14A and 14B. Galvanometer driver 15
A and 15B control the driving of the galvano scanning mirrors 14A and 14B based on signals from an image processing device described later.

The graphic pattern target 17 is disposed on the primary image plane formed by the condenser lens 16 so as to be insertable and removable by a slider 17A.

The objective lens 18 focuses the laser light passing through the graphic pattern target 17 on the sample surface 23.

The barrier filter 19 cuts the transmitted laser light which is not reflected by the dichroic mirror 13.

The condenser lens 20 is a dichroic mirror 1
The fluorescence transmitted through 3 and the barrier filter 19 is collected.

The pinhole 21 is provided at a position conjugate with the focal plane (the sample surface 23) of the objective lens 18, and allows only the fluorescence emitted from the image forming portion of the sample surface 23 to pass therethrough.

The detector 22 detects the fluorescence that has passed through the pinhole 21, converts the input fluorescence into a signal representing the light intensity, and uses the monitor (observing means) 40 to image the sample surface 23.

The computer 30 includes an image processing device 31 for imaging, a D / A converter 32, and a memory (storage means) 33. Image processing board, arithmetic circuit, general-purpose I
An image processing device 31 is constituted by an / O board or the like, and image processing software for quantifying the positional displacement of the graphic of the graphic pattern target 17 is installed in the image processing device 31.

The image processing device 31 includes a position for the control voltage of the galvano-type scanning mirrors 14A and 14B, and a change in the control voltage for the galvano-type scanning mirrors 14A and 14B.
The control signal for correcting the image shift of the graphic pattern target 17 is output to the galvanometer drivers 15A and 15B via the D / A converter 32 based on the movement amount of the galvanometer control signal. Finely adjust the offset voltages of 15a and 15b.

The computer 30 has a controller (input means) 50 for inputting the type of the dichroic mirror 13 arranged in the optical path. This controller 50
Is provided with a sensor (detection means) 25 for identifying the type of the dichroic mirror 13 arranged in the optical path.

FIG. 2 is a diagram showing a pattern example of a graphic pattern target.

On the graphic pattern target 17, a reflection pattern described later is drawn on a glass flat plate.

FIG. 2A shows an example in which one point is arranged near the center, and the shift amount in the X and Y directions is calculated based on this one point.

FIG. 2B shows an example in which grid patterns are arranged, and the shift amounts in the X and Y directions are calculated based on several points among the intersections of the grid.

FIG. 2C shows an example in which a plurality of points are arranged in a grid pattern, and the amount of displacement in the X and Y directions is calculated with reference to several of the points.

FIG. 2D shows an example in which a plurality of crosses are arranged in a grid pattern.
The shift amount in the Y direction is calculated.

The operation of the confocal laser scanning microscope having the above configuration will be described.

The excitation laser light emitted from the light source 1 passes through the excitation filter 11 and then passes through the beam expander 12.
Incident on the objective lens 1 by the beam expander 12.
It is enlarged to a size that can satisfy the pupil plane of 8.

The excitation laser light is reflected by the dichroic mirror 13, is two-dimensionally shaken by the XY scanner 14, and is focused on the sample surface 23 via the condenser lens 16, the graphic pattern target 17 and the objective lens 18. Irradiation excites a fluorescent substance in the sample to generate fluorescence.

The fluorescence is supplied to the objective lens 18, the graphic pattern target 17 and the condenser lens 1 together with the excitation laser light.
The light travels backward from 6 to the XY scanner 14 and is separated from the excitation laser light by the dichroic mirror 13.

The fluorescent light whose laser light has been cut by the barrier filter 19 is collected by the condenser lens 20, and only the fluorescent light on a predetermined slice surface of the sample surface 23 passes through the pinhole 21. Thereafter, the fluorescent light is received by the detector 22, converted into an electric signal, and displayed on the monitor 40 as an image.

FIG. 3 is a flowchart for explaining the control procedure of the microscope main body by the computer. In addition, S1
S21 indicates each step. First, a process of calculating and storing reference coordinates using the graphic pattern target 17 will be described.

The operator drives the slider 17A to insert the predetermined graphic pattern target 17 into the primary image plane (S1).

Next, the operator installs an arbitrary dichroic mirror A in the optical path (S2).

Hereinafter, S3 to S7 are control flows by the computer 30.

The computer 30 acquires an image of the graphic pattern target 17 using the dichroic mirror A (S3).

The acquired image is input to the image processing device (S4).

An arbitrary plurality of points are designated from the graphic pattern target (S5).

Position information (XY coordinates) of all designated points
Is calculated (S6).

The memory 33 uses the XY coordinates as reference coordinates.
(S7).

Next, based on the reference coordinates, dichroic mirrors B, C,...
The process of calculating and storing the shift amount of the shift will be described.

The operator sets the dichroic mirror B on the optical path (S8).

Hereinafter, S9 to S13 are control flows by the computer 30.

The computer 30 acquires an image of the graphic pattern target 17 using the dichroic mirror B (S9).

The acquired image is input to the image processing device (S10).

The same plurality of points as the dichroic mirror A are designated from the graphic pattern target 17 (S11).

The position information of all the designated points is calculated (S12).

The shift amount between the position information and the reference coordinates is calculated and stored in the memory (S13).

Steps 8 to 13 are performed for all the other dichroic mirrors 13C.

Thereafter, the slider 17A is driven to remove the graphic pattern target 17 from the primary image plane (S14).

Next, the process of placing a multi-stained sample and obtaining a multi-image thereof will be described.

The operator sets the dichroic mirror A on the optical path (S15).

The computer 30 acquires an image of the sample (S16).

Next, the operator sets the dichroic mirror B on the optical path (S17).

The computer 30 converts the amount of deviation of the dichroic mirror B from the dichroic mirror A into an analog signal by the D / A converter 32 (S18) and outputs the analog signal to the galvanometer drivers 15A and 15B (S1).
9).

Based on the analog data signal, the galvanometer drivers 15A and 15B finely adjust the offset voltage of the galvanometer control signals 15a and 15b to correct the positions of the galvanometer scanning mirrors 14A and 14B (S
20).

After the correction, an image of the sample is obtained (S2
1).

Steps 17 to 21 are performed for all the other dichroic mirrors C.

If the dichroic mirrors 13 are the same dichroic mirrors, no positional deviation occurs even if the dichroic mirrors are switched any number of times. Whenever the dichroic mirror 13 is switched, it is not necessary to correct the positional deviation using the graphic pattern target 17 and then acquire an image.

According to this embodiment, it is possible to obtain an image with no positional displacement simply by switching the dichroic mirror 13. In addition, since the optimal dichroic mirror 13 can be used for each fluorescent dye, the amount of fluorescence detected by the detector 22 can be increased, and the image does not become dark like a multi-band filter.

FIG. 4 shows a method of acquiring a multiplex image (S15 to S2).
It is a figure explaining the modification of 1).

In the above-described embodiment, an image without deviation is acquired while driving and correcting the galvanometer drivers 15A and 15B for each image. In this modified example, the galvanometer drivers 15A and 15B are not driven at the time of image acquisition. The timing for starting the image acquisition is shifted.

That is, as shown in FIG. 4, the vertical offset Vo and the horizontal offset Ho are finely adjusted based on the shift amounts stored in the memory 33, and the images obtained when each dichroic mirror is used are superimposed.

In FIG. 4, an area A1 is a scan area,
The area A2 is an image acquisition area, and the area A3 is an area after the vertical offset Vo and the horizontal offset Ho have been changed according to the shift amount stored in the memory 33.

According to this modification, the same effects as those of the above embodiment can be obtained, and the processing for superimposing images can be omitted, and the processing time can be shortened.

Further, a method of acquiring a multiplex image (S15 to S2)
A further modification of 1) will be described below.

In the above embodiment, the image deviation is corrected by driving the galvanometer drivers 15A and 15B. In this modification, the position of the stage 24 is moved to correct the image deviation.

As shown in FIG. 1, the image processing device 31
When an image is acquired by each dichroic mirror, the memory 33
The control signal is transmitted to the stage driving device 26 via the D / A converter 32 based on the deviation amount stored in the stage.

The stage driving device 26 adjusts the stage position when acquiring an image with each dichroic mirror according to the control signal.

By adjusting the stage position, the displacement of the image obtained by each dichroic mirror is accurately corrected.

According to this modification, the same effect as in the above embodiment can be exhibited.

Further, instead of calculating the shift amount using the graphic pattern target as shown in Steps 1 to 14, the error amount of the tilt angle of each dichroic mirror 13 is measured in advance, and the error is calculated from the measured value. The shift amounts are calculated, and these shift amounts are stored in the memory 33 via the controller 50.
5 to 21 may be used to obtain an image without any shift.

In the above embodiment, the graphic pattern target 17 is provided on the primary image plane. However, the graphic pattern target 17 may be provided at any position on the optical path as long as it can be held at a fixed position. It is also possible to provide it on the surface 23.

In the above embodiment, an example of a configuration in which software processing is performed using a computer has been described. However, a galvanometer driver 15A,
The driving of 15B may be controlled.

The present invention is not limited to the confocal laser scanning microscope described in the above embodiment and its modifications, but can be used for ordinary fluorescent microscopes using an ultra-high pressure mercury lamp, a halogen lamp or the like.

In this case, since there is no XY scanner, the displacement cannot be corrected by this member, but the displacement can be corrected by controlling the position of the stage.

When a CCD camera is used as the observation means, A1 shown in FIG.
Assuming that A2 and A3 are image acquisition areas to be actually acquired as images, image displacement can be corrected by shifting the image acquisition areas.

[0103]

As described above, according to the fluorescence microscope of the first aspect of the present invention, information such as an error according to the type of the light separating means, which is input to the control means by the input means, is stored. In addition, at the time of image acquisition, the observation means or the stage is controlled based on this information to correct the deviation of the acquired image, so that a bright and multiplexed image without any deviation can be obtained.

According to the fluorescence microscope of the second aspect of the present invention, information such as an error corresponding to the type of the light separating means, which is input to the control means by the input means, is stored. Since the deviation of the acquired image is corrected by controlling the scanning means based on the information, a bright and multiplexed image without deviation can be obtained.

According to the fluorescence microscope of the third aspect of the invention, the type of the light separating means can be easily identified by the detecting means for identifying the type of the light separating means arranged in the optical path.

According to the fluorescence microscope of the fourth aspect of the present invention, the offset voltage for controlling the reflection angle of the galvano-type scanning mirror is adjusted based on the shift information stored in the storage means. Analog control can accurately correct the deviation amount.

According to the fluorescence microscope of the present invention, an image acquisition area to be actually acquired as an image is selected from an area where light from a light source irradiates the sample, and the shift information stored in the storage means is selected. , The image acquisition area by the observation means is shifted, so that the image shift is corrected.

According to the fluorescence microscope of the present invention, since the stage is offset in the vertical and horizontal directions and the images are superimposed on the basis of the displacement information stored in the storage means, the image displacement is corrected. You.

According to the fluorescence microscope of the present invention, the shift amount of each light separating means is calculated from the shift amount of the graphic pattern and stored, and when acquiring an image, observation is performed based on the stored shift amount. Control means, stage or scanning means,
The displacement of the acquired image is corrected.

[Brief description of the drawings]

FIG. 1 is a block diagram of a confocal laser scanning microscope according to one embodiment of a fluorescence microscope of the present invention.

FIG. 2 is a diagram illustrating a pattern example of a graphic pattern target;

FIG. 3 is a flowchart illustrating a control procedure of a microscope main body by a computer.

FIG. 4 is a diagram illustrating a modification of the method of acquiring a multiplex image.

FIG. 5 is a diagram illustrating an example of transmission / reflection characteristics of a triple band filter.

FIG. 6 is a diagram showing the absorbance and fluorescence intensity characteristics of the fluorescent dye FITC.

FIG. 7 is a diagram showing the absorbance and fluorescence intensity characteristics of a fluorescent dye Cy3.5.

FIG. 8 is a diagram showing the absorbance and fluorescence intensity characteristics of the fluorescent dye Cy5.

[Explanation of symbols]

 Reference Signs List 1 laser light source 13 dichroic mirror (light separating means) 14 XY scanner (scanning means) 14A, 14B galvano-type scanning mirror 18 objective lens 25 sensor (detecting means) 30 computer (control means) 33 memory (storage means) 40 monitor (observation) Means) 50 controller (input means)

Claims (7)

[Claims] 1. A light source that emits excitation light, a stage on which a sample is mounted, and a plurality of switches provided in a light path so as to be switchable and reflect excitation light from the light source and transmit fluorescence from the sample. In a fluorescence microscope having a light separating unit and an observation unit for obtaining an image of fluorescence transmitted through the light separating unit, an input unit for inputting information regarding a type of the light separating unit arranged in the optical path at the time of image acquisition. A storage unit for storing image shift information at the time of image acquisition in each of the light separating units; and reading out the image shift information stored in the storage unit based on information from the input unit; Control means for controlling the observation means or the stage so that images obtained by the separation means are superimposed on each other. 2. A light source that emits an excitation laser beam, a plurality of light separating means that reflect the excitation laser beam, transmit fluorescence, and are switchably provided in an optical path; A fluorescence microscope comprising: a scanning unit that performs two-dimensional scanning on the scanning unit; an objective lens disposed between the scanning unit and the sample; and an observation unit that obtains an image of fluorescence transmitted through the light separation unit. Input means for inputting information about the type of the light separating means arranged in the light path; storage means for storing image shift information at the time of image acquisition in each of the light separating means; and information based on the information from the input means. Read out the image shift information stored in the storage means, and control the scanning means so that images obtained by different types of light separation means are superimposed. Fluorescence microscope, characterized in that a control means. 3. The fluorescence microscope according to claim 1, wherein the input unit includes a detecting unit that identifies a type of the light separating unit disposed on the optical path. 4. The scanning unit includes a galvano-type scanning mirror, and the control unit adjusts an offset voltage for controlling a reflection angle of the galvano-type scanning mirror based on the shift information stored in the storage unit. 3. The method according to claim 2, wherein
The fluorescence microscope according to 1. 5. The observation means selects an image acquisition area from an area where light from the light source irradiates the sample, and the control means, based on shift information stored in the storage means, 2. The fluorescence microscope according to claim 1, wherein a timing at which an image acquisition by the observation means is started is controlled. 6. The fluorescence microscope according to claim 1, wherein said control means controls the position of said stage based on shift information stored in said storage means. 7. The apparatus according to claim 1, further comprising a graphic pattern arranged on a primary image plane of the objective lens, wherein the observation unit obtains an image of the graphic pattern for each of the light separating units, and obtains one of the obtained images. 7. A shift amount of another image is calculated on the basis of one of the images, and is stored in the storage unit as shift information.
The fluorescence microscope according to any one of the above.