JP2004021206A - Light source adjusting device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a light source adjusting device capable of quantitatively adjusting a light source. <P>SOLUTION: The light source adjusting device has a Koehler illumination system having a collector lens 4 projecting the picture of the light source 2 to the position of the pupil 10a of an objective 10, and the lens 4 is moved in an optical axis direction by a focusing part 3. A CCD camera 12 photographs with light from a sample 11 irradiated with the light from the light source 2 through the lens 4, and light intensity is fetched from the taken picture data so as to arithmetically calculate the irregularity of light quantity in an arithmetic part 13. Then, the obtained result is displayed on a display part 14. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a light source adjustment device applied to an illumination optical system such as a microscope.
[0002]
[Prior art]
In Koehler illumination, the image of the light source is created on the pupil of the objective lens, and the image of the aperture of the light source (field stop) is formed on the specimen. It is bright, uniform, and fully utilizes the performance of the objective lens. It is a lighting method that can be used.
[0003]
By the way, when observing a sample using such Koehler illumination method, it is necessary to adjust the focus of the light source.
[0004]
Here, the focus adjustment of the light source is to adjust the position of the collector lens so as to obtain a state in which the image of the light source is projected on the pupil of the objective lens. Such a focus adjustment is performed accurately. This makes it possible to observe the specimen in the brightest and even state.
[0005]
As described above, the focus adjustment of the light source is an important task, but it is also a difficult task. Therefore, various adjustment methods have been conventionally considered.
[0006]
Japanese Patent Laying-Open No. 11-72713 discloses an example of a focus adjustment of a light source. In a housing having a built-in lamp and a collector lens, a focus adjustment mechanism for adjusting the focus of the lamp by moving the collector lens in the optical axis direction is provided. A scale indicating the focus position is provided on the adjustment knob of the focus adjustment mechanism, and the adjustment knob can be rotated so that the focus can be adjusted by simply adjusting the pointer to the scale.
[0007]
[Problems to be solved by the invention]
However, in such a configuration, the positional relationship between the scale indicating the focus position and the collector lens must be accurately determined in advance. However, the determination of the positional relationship here involves human work. In addition, since this work involves the subjectivity of the individual worker, variations due to differences in the adjustment of the workers are likely to occur, and stable adjustment is difficult.
[0008]
Further, even if the positional relationship between the scale and the collector lens is calculated from design values or the like, individual differences between lamps and lamp houses cannot be covered. Furthermore, even if a scale that accurately indicates the focus position of the collector lens is provided, if the temperature of the lamp house rises and the dimensions change due to thermal expansion or the like, the scale and the position of the collector lens may be changed. The relationship changes, and there arises a problem that accurate adjustment cannot be performed only by adjusting the pointer to the scale using the adjustment knob.
[0009]
The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a light source adjustment device capable of quantitatively adjusting a light source.
[0010]
[Means for Solving the Problems]
The invention according to claim 1 includes a light source, a Koehler illumination system having a collector lens for condensing light from the light source, a collector lens moving means for moving the collector lens in the optical axis direction, and the collector lens. A sample irradiated with light from the light source, imaging means for photographing light from the sample, arithmetic means for extracting light intensity from image data acquired by the imaging means, and calculating light intensity unevenness, Display means for displaying the calculation result of the means.
[0011]
According to a second aspect of the present invention, in the first aspect, the sample is made of a fluorescent member that emits uniform fluorescence.
[0012]
The invention according to claim 3, wherein a Koehler illumination system having a light source, a collector lens for condensing light from the light source, a collector lens electric driving means for electric driving of the collector lens in the optical axis direction, and the collector lens A sample irradiated with light from the light source through, an imaging unit that captures light from the sample, and a calculation unit that calculates light intensity unevenness by extracting light intensity from image data acquired by the imaging unit, And controlling the collector lens electric drive unit based on the calculation result of the calculation unit to set the optimum position of the collector lens.
[0013]
As a result, according to the present invention, the adjustment of the light source can be performed quantitatively using the calculation result of the calculated light amount unevenness.
[0014]
Further, according to the present invention, the positioning of the collector lens can be automatically performed based on the calculation result of the uneven light amount, and the light source can be automatically adjusted.
[0015]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[0016]
(First Embodiment)
FIG. 1 shows a schematic configuration of an array reader using a one-box type fluorescence microscope used for analysis of a DNA chip to which the present invention is applied.
[0017]
In the figure, one lamp house 1 houses a light source 2. In this case, a mercury lamp, a xenon lamp, a metal halide lamp, or the like is used as the light source 2.
[0018]
The lamp house 1 is provided with a focus adjustment unit 3 as a collector lens moving unit. The focus adjustment unit 3 is provided with a cylindrical lens holding unit 3a, and a collector lens 4 is held in a hollow portion of the lens holding unit 3a.
[0019]
The lens holding section 3a has a knob 3b. When the knob 3b is rotated, the collector lens 4 moves in the optical axis direction in accordance with the rotation, and the movement enables the focus adjustment of the light source 2. In this case, for example, an eccentric cam is attached to the knob 3b, and the relationship between the rotation of the knob 3b and the movement of the collector lens 4 in the optical axis direction is such that the knob 3b rotates once (360 °). Then, the collector lens 4 is moved forward by a predetermined distance and is moved backward by the same distance.
[0020]
The light emitted from the light source 2 is converted into parallel light through the collector lens 4 and enters the excitation filter 7 via the shutter 5, the relay lens system 6, the AS (aperture stop) 16, and the FS (field stop) 17. Is done.
[0021]
The excitation filter 7 extracts only excitation light for exciting the fluorescence of the sample 11. The excitation light extracted by the excitation filter 7 is reflected by the dichroic mirror 8 and irradiates the sample 11 via the objective lens 10.
[0022]
Further, the fluorescence emitted from the sample 11 by the irradiation of the excitation light passes through the dichroic mirror 8, and after unnecessary wavelength components are removed by the absorption filter 9, the CCD as an imaging means is passed through the imaging lens 18. An image is formed on the imaging surface of the camera 12 and imaged.
[0023]
Here, as the CCD camera 12, for example, CooISNAP-Pro MONOCROME CAMERA manufactured by Roper Scientific is used. Of course, the CCD camera 12 is not limited to this, and may be any imaging means.
[0024]
The image data of the CCD camera 12 is taken into an operation unit 13 as operation means. The calculation unit 13 calculates the light intensity unevenness by taking in the light intensity from the image data, displays the result on the display unit 14 of the display means, and evaluates the optimal light source adjustment.
[0025]
Here, an algorithm for capturing the light intensity and calculating the light intensity unevenness in the calculation unit 13 will be briefly described.
[0026]
Now, suppose that the average intensity value Iave and the light intensity unevenness ΔI of the image data captured by the CCD camera 12 are acquired and these two values are used to evaluate the optimal light source adjustment. Attention is focused on 20 × 20 = 400 points B in the image A shown in FIG. One point here is a circular area of 20 pixels (about 100 μm) in diameter.
[0027]
Then, the average value of the luminance values in each of the points B is obtained, and the average value is used as the light intensity value in the unit of the point B, and the average of the light intensity values of the 400 points B is obtained as the average intensity value Iave.
[0028]
On the other hand, the light amount unevenness ΔI is obtained from the following.
[0029]
ΔI = {(Imax−Imin) / Iave} × 100 [%] (1)
Here, Iave is an average light amount value in a certain range, Imax is a highest light amount value in a certain range, and Imin is a minimum light amount value in a certain range.
[0030]
Equation (1) indicates that the smaller the value of ΔI, the more uniform and uniform. Therefore, it is possible to evaluate the uneven light amount of the illumination light by calculating the uneven light amount ΔI by substituting the average intensity value Iave obtained from the 400 points B into the equation (1). it can. In addition, such an evaluation of the light amount unevenness is performed at a large number of positions by moving the collector lens 4 along the optical axis, and finding the point that is evaluated the best among them, thereby performing the optimal light source adjustment. Is possible.
[0031]
As an example, a graph shown in FIG. 3 is obtained by plotting the unevenness in light amount ΔI calculated by the experiment and the average intensity value Iave. Here, the lens holding unit 3a of the focus adjustment unit 3 is rotated by 5 ° by 360 ° by operating the knob unit 3b, and images captured by the CCD camera 12 at each angle are acquired, and the above described (1) The average intensity value Iave (a in the figure) and the light amount unevenness ΔI (b in the figure) are obtained by using the equations. In the graph of FIG. 3, the horizontal axis represents the rotation angle of the knob 3b of the focus adjustment unit 3. In addition, according to the rotation angle of the knob 3b at this time, the collector lens 4 moves while changing the moving distance 1 in the front-rear direction along the optical axis as shown in FIG.
[0032]
In this graph, the condition of Koehler illumination is that there is no unevenness and the brightness is high, so that a point where the unevenness of the light amount ΔI is small and the average intensity value Iave is large is found. The position of the collector lens 4 in a state where the image of the light source 2 is projected on the pupil 10a of the objective lens 10 can be obtained.
[0033]
In addition, the calculation unit 13 creates the following two graphs, which serve as operation references for actually rotating the knob 3b of the focus adjustment unit 3, using the results, and displays the results on the display unit. 14 is displayed.
[0034]
FIG. 4 is a graph showing an intensity profile. This graph shows the luminance distribution in one direction arbitrarily set from the image taken in from the CCD camera 12, and the graph is used to visually display the uniformity of the luminance distribution as a numerical value. FIG. 5 shows the relationship between the average intensity value Iave and the uneven light amount ΔI calculated and displayed as evaluation points. From this calculation result, when the relationship between the average intensity value Iave and the uneven light amount ΔI shows the minimum value (m in the drawing), it indicates that the uneven light amount is the smallest.
[0035]
An example in which light source adjustment is actually performed in such a configuration will be described.
[0036]
In this case, a fluorescent member, for example, a fluorescent substrate that emits uniform fluorescent light is used as the sample 11. For this fluorescent substrate, for example, Sumipex 651 (trade name) manufactured by Sumitomo Chemical is used.
[0037]
The operation procedure from this state will be described with reference to the flowchart shown in FIG.
[0038]
First, in step 601, light source adjustment is started, and in step 602, the shutter 5 is opened, and the illumination light emitted from the light source 2 and converted into parallel light through the collector lens 4 is guided to the optical path. This light is reflected by a dichroic mirror 8 as excitation light via an excitation filter 7 and is applied to a sample 11 via an objective lens 10.
[0039]
The fluorescent component emitted from the sample 11 by the irradiation of the excitation light passes through the dichroic mirror 8 and is captured as a moving image of the surface of the sample 11 by the CCD camera 12 via the absorption filter 9 (step 603).
[0040]
Next, in step 604, the lens holding unit 3a of the focus adjustment unit 3 is rotated by a predetermined angle (for example, about 5 °) by operating the knob unit 3b, and the collector lens 4 is moved by a predetermined distance along the optical axis.
[0041]
Then, even in this state, the light passing through the collector lens 4 is applied to the sample 11 via the excitation filter 7, the fluorescence emitted from the sample 11 is acquired by the CCD camera 12, and transmitted to the arithmetic unit 13 as image data. I do. The arithmetic unit 13 calculates the average intensity value Iave and the light intensity unevenness ΔI from the image data, generates the above-mentioned intensity profile and evaluation points, and temporarily stores them (step 606).
[0042]
Next, it is determined whether or not the adjustment has been completed in step 607. If the adjustment has not been completed, the process returns to step 604, the knob 3b of the focus adjustment unit 3 is rotated again by a predetermined angle, and the collector lens 4 is moved along the optical axis. Is further moved a predetermined distance.
[0043]
Hereinafter, similarly, the knob 3b of the focus adjustment unit 3 is rotated once by a predetermined angle (360 °), and the collector lens 4 is advanced by a predetermined distance along the optical axis, and is retracted by this advanced distance. Then, the image data at each position of the collector lens 4 is acquired by the CCD camera 12, the arithmetic unit 13 calculates the average intensity value Iave and the light amount unevenness ΔI, and stores the respective intensity profiles and evaluation points.
[0044]
After that, the evaluation points thus obtained are displayed on the display unit 14 as a graph as shown in FIG.
[0045]
The operator refers to this graph to judge that the point m at which the relationship between the average intensity value Iave and the uneven light amount ΔI shows the minimum value is in the state where the uneven light amount is the smallest, and based on the result of this determination, the collector lens 4 is determined in the optical axis direction. Further, the uniformity of the luminance distribution at this position is visually confirmed by a graph of the intensity profile in FIG. Thereby, the optimal light source adjustment is completed. That is, in this state, the position of the collector lens 4 is adjusted so that the image of the light source 2 is projected on the pupil 10a of the objective lens 10.
[0046]
If it is determined in step 607 that the adjustment has been completed, the process proceeds to step 608, where the shutter 5 is closed, and in step 609, the light source adjustment ends.
[0047]
After that, if the DNA chip 15 is arranged as the sample 11 instead of the fluorescent substrate, the observation result by Koehler illumination is obtained by the CCD camera 12.
[0048]
Accordingly, with this configuration, the state of the smallest light quantity unevenness is determined from the graph of the evaluation points representing the relationship between the average intensity value Iave and the light quantity unevenness ΔI when the collector lens 4 is moved along the optical axis. Only by doing so, the collector lens 4 can be adjusted to the optimum position on the optical axis, so that the light source can be quantitatively adjusted. Accordingly, the operation for adjusting the light source is extremely simple, and even if the adjustment is performed for the first time or an inexperienced person, the optimal state of the illumination can be adjusted accurately.
[0049]
In the above description, a one-box type array reader using a fluorescence microscope has been described. However, the configuration shown here is an example, and an ordinary fluorescence microscope, a bright-field microscope, or the like requires a similar light source adjustment device. Anything that can be applied is applicable. Further, as a light source to be used, for example, a mercury lamp, a xenon lamp, a metal halide lamp, or the like can be implemented, whereby the characteristics of a filter to be used can be arbitrarily selected.
[0050]
In the above description, the lens holding unit 3a of the focus adjustment unit 3 is rotated once by the knob 3b so as to deepen the optimum position of the collector lens 4. However, before the experiment, the user needs the minimum intensity value line. Is set, the position where the light intensity unevenness ΔI shows the minimum value at the position of the collector lens 4 where the intensity value is equal to or more can be determined to be optimal. This makes it possible to prevent the case where the minimum intensity value is erroneously selected when two minimum values are possessed as shown in FIG.
[0051]
Further, in the above-described embodiment, 400 points were selected as the average luminance value. However, the number of points to be selected is not limited to 400 points. It is also possible to reduce the number of points and to increase the number of points selected to further increase the accuracy.
[0052]
Furthermore, as the optimum state, the point where the light quantity unevenness is minimized (the point where ΔI becomes the smallest) is selected. Even if it gets bigger, it is considered that there are two cases where the user wants to use it brighter and use it. In this case, it is necessary to set a user for weighting of luminance priority and unevenness priority. In the above-described embodiment, this selection is possible, and setting of an optimal state for the user can be arbitrarily performed.
[0053]
Furthermore, in the above-described embodiment, the end of the adjustment is visually determined in a state where the graph of FIG. 5 shows the minimum value, but when the minimum value is shown, the sound is output from the calculation unit 13. The same effect can be obtained even if the method of (1) is adopted. This has an effect that, even when the display unit 14 is far away and is difficult to see, the knob 3b may be operated by relying on the sound, and the adjustment can be easily performed.
[0054]
Further, even if the knob 3b is inadvertently moved after the focus adjustment, the adjustment can be made again by searching for the minimum value while looking at the graph of FIG. 5, and the correction can be easily made. Further, since the collector lens 4 can be returned to the same position by this method, the same state as that after the initial focus adjustment can be easily reproduced.
[0055]
(Second embodiment)
Next, a second embodiment of the present invention will be described.
[0056]
FIG. 7 shows a schematic configuration of the second embodiment, and the same parts as those in FIG. 1 are denoted by the same reference numerals.
[0057]
In this case, FIG. 7 is different from the first embodiment shown in FIG. 1 in that the light emitted from the light source 2 passes through the collector lens 4 in parallel with the transmission type microscope. The light is converted into light, and enters the excitation filter 7 via the shutter 5. The excitation light extracted by the excitation filter 7 is reflected by the mirror 21 through the FS 17 and is irradiated on the sample 11 through the window lens 19, the AS 16 and the condenser lens 20, and the transmitted light from the sample 11 is The image is captured by the CCD camera 12 via the absorption filter 9 and the imaging lens 18.
[0058]
In this way, the same effects as those of the first embodiment can be expected, and the light source adjustment device of the present invention can be applied not only to the epi-illumination type fluorescence microscope but also to the transmission type microscope.
[0059]
(Third embodiment)
Next, a third embodiment of the present invention will be described.
[0060]
FIG. 8 shows a schematic configuration of the third embodiment, and the same parts as those in FIG. 1 are denoted by the same reference numerals.
[0061]
In this case, the focus adjustment unit 3 provided in the lamp house 1 is configured as a collector lens electric drive unit that electrically drives the rotation of the lens holding unit 3a, and a motor 31 is provided instead of the knob.
[0062]
The arithmetic unit 13 is connected to the motor 31. The arithmetic unit 13 sends a control signal to the motor 31 to control the movement of the collector lens 4 to perform focus adjustment.
[0063]
The operation procedure with such a configuration will be described with reference to the flowchart shown in FIG.
[0064]
First, in step 901, light source adjustment is started. In step 902, the shutter 5 is opened, and the illumination light emitted from the light source 2 and converted into parallel light through the collector lens 4 is guided to the optical path.
[0065]
In step 903, the control unit 13 sends a control signal to the motor 31, rotates the lens holding unit 3a by a predetermined angle, for example, 5 °, and moves the collector lens 4. In step 904, the image of the sample 11 obtained by the illumination light at that position is captured by the CCD camera 12. In step 905, the arithmetic unit 13 calculates the average intensity value Iave and the light amount unevenness ΔI from the captured image by the method described in the first embodiment. To save temporarily.
[0066]
Next, in step 906, it is determined whether the movement of the collector lens 4 has reached the entire area. If the movement has not been completed, the process returns to step 903, where a control signal is sent from the calculation unit 13 to the motor 31, and the focus adjustment unit The collector lens 4 is moved by rotating the lens holder 3a of the third lens 3 by a predetermined angle.
[0067]
In the same manner, the lens holding unit 3a of the focus adjustment unit 3 is rotated once (360 °) by a predetermined angle by the motor 31 to advance the collector lens 4 by a predetermined distance along the optical axis, and to retract by the advance distance. Let it. Then, the image data at each position of the collector lens 4 is acquired by the CCD camera 12, the arithmetic unit 13 calculates the average intensity value Iave and the light amount unevenness ΔI, and stores them in the arithmetic unit 13.
[0068]
Thereafter, if it is determined in step 906 that the movement of the collector lens 4 has reached the entire area, the process proceeds to step 907, where the shutter 5 is closed, and the image acquisition ends.
[0069]
Next, in step 908, in the same manner as described in the first embodiment, the relationship between the average intensity value Iave acquired by moving the entire region of the collector lens 4 and the light amount unevenness ΔI is determined, and these relationships are determined. Find the point at which shows the minimum value.
[0070]
In step 909, based on the minimum value at this time, the motor 31 rotates the lens holding unit 3a of the focus adjustment unit 3 to move the collector lens 4 to the optimum position, and ends the light source adjustment in step 910. . That is, in this state, the position of the collector lens 4 is adjusted so that the image of the light source 2 is projected on the pupil 10a of the objective lens 10.
[0071]
Therefore, in this manner, the positioning of the collector lens 4 can be automatically performed without human intervention, and an optimal illumination state can be obtained. This makes it impossible to manually operate the lens holding unit 3a of the focus adjustment unit 3, for example, when the knob of the lens holding unit 3a is not reachable, or when the knob enters the one-box type device. In such a case, the light source can be easily adjusted.
[0072]
Further, it is possible to easily cope with a change in the illumination state due to a change in the light source 2 or a change in the mirror unit (one having the excitation filter 7, the dichroic mirror 8, the absorption filter 9, etc.). Therefore, by setting up a measurement routine that periodically re-adjusts the measurement, it is possible to always perform the measurement under the optimal and constant illumination condition.
[0073]
In the above-described second embodiment, the position where the relationship between the average intensity value Iave and the light intensity unevenness ΔI shows the minimum value is searched, but the user needs to set the necessary minimum intensity value in advance. Therefore, it is also possible to realize a method in which the position where the intensity unevenness ΔI has the minimum value at the position of the collector lens 4 at which the intensity value is equal to or more than the intensity value is determined to be optimal. In addition to this, by setting the minimum intensity value, even if there are two minimum values, it is possible to reliably exclude one of the states.
[0074]
In addition, the present invention is not limited to the above-described embodiment, and can be variously modified in an implementation stage without departing from the spirit of the invention. In the above-described embodiment, at the time of adjusting the light source, a plastic material is used as the fluorescent substrate that emits uniform fluorescent light as the sample 11, but instead of this, fluorescent glass, opal glass, ceramic phosphor, or the like may be used. It is possible. They have good durability, little discoloration, and little change in performance, and can be used for a long time. In particular, fluorescent glass is more preferable because the fluorescent light emission is uniform in any part. Further, since the excitation wavelength can be selected by the fluorescent glass, it is a very good point that the light source can be adjusted in consideration of the characteristics of the filter. Opal glass is a general material and is preferable because it is easily available and inexpensive.
[0075]
Furthermore, the above embodiments include inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent features. For example, even if some components are deleted from all the components shown in the embodiments, the problem described in the column of the problem to be solved by the invention can be solved, and the problem described in the column of the effect of the invention can be solved. In the case where the effect described above is obtained, a configuration from which this configuration requirement is deleted can be extracted as an invention.
[0076]
【The invention's effect】
As described above, according to the present invention, it is possible to provide a light source adjustment device capable of quantitatively adjusting a light source.
[Brief description of the drawings]
FIG. 1 is a diagram showing a schematic configuration of a fluorescence microscope applied to a first embodiment of the present invention.
FIG. 2 is an image in which measurement points for extracting light intensity values from an image acquired in the first embodiment are arranged.
FIG. 3 is a diagram illustrating an example of a measurement result of a relationship between uneven light intensity and an average intensity value with respect to a rotation angle of a knob according to the first embodiment.
FIG. 4 is a schematic diagram of a graph displaying an intensity profile obtained from an image obtained in the first embodiment.
FIG. 5 is a diagram illustrating an example of a measurement result of a relationship between light intensity unevenness and an average intensity value from a calculation result according to the first embodiment.
FIG. 6 is a flowchart for explaining the operation of the first embodiment.
FIG. 7 is a diagram showing a schematic configuration of a second embodiment of the present invention.
FIG. 8 is a diagram showing a schematic configuration of a third embodiment of the present invention.
FIG. 9 is a flowchart for explaining the operation of the third embodiment.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Lamp house 2 ... Light source 3 ... Focus adjustment part 3a ... Lens holding part 4 ... Collector lens 5 ... Shutter 6 ... Relay lens system 7 ... Excitation filter 8 ... Dichroic mirror 9 ... Absorption filter 10 ... Objective lens 11 ... Sample 12 ... CCD camera 13 Operation unit 14 Display unit 15 DNA chip 21 Mirror 31 Motor

Claims (3)

A light source,
A Koehler illumination system having a collector lens for condensing light from the light source,
Collector lens moving means for moving the collector lens in the optical axis direction,
A sample irradiated with light from the light source through the collector lens,
Imaging means for capturing light from the sample,
An arithmetic unit for extracting light intensity from the image data acquired by the imaging unit and calculating the light intensity unevenness;
Display means for displaying a result of the calculation by the calculation means. The light source adjusting device according to claim 1, wherein the sample is made of a fluorescent member that emits uniform fluorescent light. A light source,
A Koehler illumination system having a collector lens for condensing light from the light source,
Collector lens electric drive means for electrically driving the collector lens in the optical axis direction,
A sample irradiated with light from the light source through the collector lens,
Imaging means for capturing light from the sample,
Computing means for extracting light intensity from the image data obtained by the imaging means and calculating light quantity unevenness,
A light source adjustment device, wherein the collector lens electric drive unit is controlled based on a calculation result of the calculation unit to set an optimum position of the collector lens.

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