JP2006171024A - Multipoint fluorescence spectrophotometric microscope and multipoint fluorescence spectrophotometric method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a multipoint fluorescence spectrophotometric microscope and a multipoint fluorescence spectrophotometric method capable of easily setting an arbitrary measurement target region on a specimen and obtaining spectral information of these measurement target regions with high accuracy.
A DMD 3 is arranged at a position optically conjugate with a focal position of an objective lens 1 for condensing light from an illumination optical system that generates light from a light source on the sample S, and light generated from the sample S. The ROI on the specimen S is set from the imaging screen of the CCD camera 13 that picks up the image, the reflection pattern of the DMD 3 is controlled according to the set ROI, and the light generated from the region corresponding to the ROI on the specimen S is Obtained by wavelength dispersion by the grating 18 and the line photosensor array 19.
[Selection] Figure 1

Description

  The present invention relates to a multipoint fluorescence spectrophotometric microscope and a multipoint fluorescence spectrophotometric method for spectrophotometrically measuring fluorescence of a plurality of measurement target regions in a specimen.

  Recently, in order to confirm the active state of cells, expression of specific genes, and the like, a method of detecting fluorescence emitted from intracellular substances using a microscope has been used. In such a fluorescence detection method, in particular, it is required to select a plurality of cells in a specimen or to individually analyze fluorescence spectral wavelength characteristics with higher speed and higher accuracy.

  From such a background, a target region (Region Of Interest: hereinafter referred to as “ROI”) is arbitrarily set as a measurement target region to be observed or measured in the specimen, and a specific wavelength is set for these ROIs. As a so-called optical modulation means, for example, only a small deflection element such as a digital micro-mirror device (hereinafter referred to as “DMD”) has been put into practical use. Here, the DMD has a micromirror of about 16 μm square as one pixel, and the tilt angle can be controlled for each pixel piece.

As a microscope using such a DMD, as disclosed in Patent Document 1, a DMD is arranged at a position optically conjugate with a sample surface in an illumination optical system, and a pattern selectively set by the DMD is used. The illumination light is projected onto the specimen surface, and the light from the specimen illuminated by the DMD pattern is disposed at a position optically conjugate with the specimen surface in the observation optical system and transmits the illumination pattern having a shape conjugate with the DMD. A DMD is arranged at a position optically conjugate with the sample surface in the illumination optical system as disclosed in Patent Document 2 and selectively entered by the DMD. The illumination light of the set pattern is projected onto the sample surface, and the light from the sample illuminated by the DMD pattern is converted into the sample image at a position optically conjugate with the sample surface in the observation optical system. Those to be observed Te has been considered.
Japanese Patent Laid-Open No. 11-249023 JP 2003-107361 A

  However, in Patent Document 1, the DMD is disposed at a position optically conjugate with the sample surface in the illumination optical system, and a pattern having a shape conjugate with the DMD is formed at a position optically conjugate with the sample surface in the observation optical system. In order to construct a confocal spectroscopic microscope with this pattern (slit shape in the reference) as a confocal stop, to obtain an ROI image arbitrarily set on the sample, the pattern is scanned sequentially. There is a problem that it is necessary to make a three-dimensional image, and it takes time and labor to acquire the ROI image.

  Further, in Patent Document 2, a DMD is disposed at a position optically conjugate with a sample surface in an illumination optical system, and an ROI arbitrarily set on the sample by a DMD pattern is two-dimensionally illuminated. Although a two-dimensional image can be acquired in real time, nothing is disclosed about spectral detection of data acquired as a two-dimensional image in this way.

  The present invention has been made in view of the above circumstances, and can easily set arbitrary measurement target regions on a specimen, and can efficiently acquire spectral information of these measurement target regions and multipoint fluorescence spectrophotometric microscopes and multipoints An object is to provide a fluorescence spectrophotometric method.

  The invention described in claim 1 is a light source, an illumination optical system that irradiates the specimen with light from the light source, an objective lens that forms the specimen image, and an optical conjugate with the specimen in the illumination optical system. A micro deflection element provided at a position, an imaging means for capturing a specimen image formed by the objective lens, a measurement target area setting means for setting a measurement target area on the specimen from a captured image of the imaging means, Control means for controlling the light deflection pattern of the micro deflection element in accordance with the measurement target area set by the measurement target area setting means, and obtaining the wavelength-dispersed light generated from the measurement target area on the sample. And a spectral detection means.

  The invention according to claim 2 is a light source, an illumination optical system that generates light from the light source, an objective lens that condenses light from the illumination optical system on a specimen, and the objective lens in the illumination optical system. A micro-deflection element provided at a position optically conjugate with the focal position of the lens, an imaging means for imaging light generated from the specimen, and a measurement for setting a measurement target region on the specimen from an image captured by the imaging means Target region setting means, control means for controlling the light deflection pattern of the micro deflection element according to the measurement target region set by the measurement target region setting means, and light generated from the measurement target region on the specimen And a spectral detection means for obtaining by wavelength dispersion.

  The invention according to claim 3 is a light source, an illumination optical system that generates light from the light source, an objective lens that collects light from the illumination optical system on a specimen, and the objective lens in the illumination optical system. A position that is optically conjugate with the focal position of the objective lens in the first observation optical system having a micro deflection element provided at a position optically conjugate with the focal position of the lens and an imaging lens passing through the objective lens An imaging means for capturing a projected image of the specimen, a fiber for transmitting the projected image of the specimen with an incident surface disposed at a position optically conjugate with the imaging means, and light emitted from the fiber Spectral detection means obtained by wavelength dispersion, measurement target area setting means for setting a measurement target area on the sample from the imaging screen of the imaging means, and measurement target areas set by the measurement target area setting means. The controls the deflection pattern of the fine deflecting device is characterized by comprising a control means for processing the acquired data of the spectroscopic detection means Te.

  According to a fourth aspect of the present invention, in the invention according to any one of the first to third aspects, the measurement target region setting means sets an arbitrary region emitting light from the imaging screen as the measurement target region. It is characterized by.

  According to a fifth aspect of the invention, in the invention according to any one of the first to third aspects, the measurement target region setting means is based on binarized data of luminance values of light emitted from the imaging screen. It is characterized by setting a measurement target region.

  According to a sixth aspect of the present invention, in the second observation optical system according to any one of the first to fifth aspects, the incident surface of the fiber includes another imaging lens that passes through the objective lens. It is characterized by being arranged at a position optically conjugate with the imaging means.

  The invention according to claim 7 is a first step of acquiring a captured image of the entire field of the specimen irradiated with light from the light source via the illumination optical system, and the captured image acquired in the first step. A second step of setting a measurement target region on the sample; and a measurement optical region conjugate with the sample of the illumination optical system according to the measurement target region set in the second step. A third step for controlling the deflection pattern of the micro deflection element, and a measurement target region on the specimen irradiated with light through the micro deflection element whose deflection pattern is controlled in the third step. And a fourth step of acquiring the light to be dispersed by wavelength dispersion.

  According to the present invention, it is possible to provide a multipoint fluorescence spectrophotometric microscope that can easily set an arbitrary measurement target region on a specimen and can efficiently acquire spectral information of these measurement target regions.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

(First embodiment)
FIG. 1 shows a schematic configuration of a multipoint fluorescence spectrophotometric microscope according to the first embodiment of the present invention.

  In the figure, S is a sample, and the objective lens 1 is disposed in the vicinity of the sample S. The objective lens 1 condenses illumination light on the specimen S, and makes the light incident from the focal position into substantially parallel light for observation.

  A mirror 2 is disposed on the observation optical axis A of the objective lens 1. The mirror 2 is at the intersection with the illumination optical axis B1 and reflects the illumination light toward the objective lens 1 side.

  In this case, the mirror 2 has a characteristic of reflecting a transmission wavelength region of an excitation filter 7 to be described later and transmitting a transmission wavelength region of the absorption filter 8. For example, the mirror 2 has a reflectivity regardless of a long-pass dichroic mirror or a wavelength. A half mirror that is substantially constant is used.

  A DMD 3 is disposed on the illumination optical axis B1 at the intersection with the illumination optical axis B2. In this case, the DMD 3 is in a positional relationship optically conjugate with the focal position of the objective lens 1.

  On the illumination optical axis B1 between the DMD 3 and the mirror 2, a projection lens 4 and an excitation filter 7 constituting an illumination optical system are disposed. The projection lens 4 is for projecting the image of the DMD 3 onto the focal plane of the objective lens 1. The excitation filter 7 is a band-pass filter that transmits illumination light in a predetermined wavelength range, and is inserted into the illumination optical path when performing fluorescence observation.

  A light source 5 is disposed on the illumination optical axis B2 incident on the DMD 3. Further, a collector lens 6 is disposed on the illumination optical axis B2 between the light source 5 and the DMD 3 so as to make light emitted from the light source 5 substantially parallel.

  In this case, the DMD 3 reflects the light incident from the direction of the illumination optical axis B2 in the direction of the illumination optical axis B1 with the micromirrors constituting the reflection (deflection) pattern turned on, and turns off the illumination light in the off state. Light incident from the direction of the axis B2 is reflected in another direction away from the illumination optical axis B1. Thereby, the light emitted from the light source 5 is selectively guided in the direction of the illumination optical axis B1 by the reflection pattern of the DMD 3 and projected onto the sample S as illumination light through the projection lens 4, the mirror 2, and the objective lens 1. .

  An absorption filter 8, an imaging lens 9, and a first observation optical path branching unit 10 are disposed on the transmission optical path side viewed from the sample S of the mirror 2, that is, on the extension of the observation optical axis A.

  The absorption filter 8 is a long-pass filter that transmits only a wavelength range longer than the transmission wavelength range of the excitation filter 7 and is inserted into the optical path together with the excitation filter 7 when performing fluorescence observation. The first observation light path branching unit 10 enables switching of a plurality of prisms or insertion / removal of a single mirror (switching of a plurality of prisms in FIG. 1). The observation optical axis A1 which is transmitted and the observation optical axis A2 which is reflected are branched into two.

  An eyepiece lens 11 is disposed on the observation optical axis A1. The eyepiece 11 is disposed at the image forming position of the image forming lens 9 so that the observation image of the sample S can be visually observed.

  On the extension of the observation optical axis A2, the second observation optical path branching portion 12 is disposed. The second observation optical path branching section 12 enables switching of a plurality of prisms or insertion / removal of a single mirror (in FIG. 1, insertion / removal of a single mirror), and has advanced the observation optical axis A2. The light is branched into an observation optical axis A3 that transmits light and an observation optical axis A4 that reflects light.

  On the observation optical axis A3, a CCD camera 13 as an imaging means is arranged. In the CCD camera 13, an imaging surface (not shown) is positioned at the focal position of the imaging lens 9 (a position optically conjugate with the focal position of the objective lens 1), and the projection image of the sample S is captured. .

  On the observation optical axis A4, the relay lens 14, the reduction lens 15, and the incident surface 16a of the optical fiber 16 are arranged. The relay lens 14 converts the observation light emitted after the projection image of the sample S is formed into a substantially parallel light at the focal position of the imaging lens 9 (a position optically conjugate with the focal position of the objective lens 1). is there. The reduction lens 15 collects the parallel light from the relay lens 14 and forms a projection image of the sample S at a reduction magnification. In the optical fiber 16, the incident surface 16 a is disposed at the focal position of the reduction lens 15, and a projection image of the sample S formed with a reduction magnification is incident thereon.

  On the optical path from the emission surface 16b of the fiber 16, a collimating lens 17, a grating 18 constituting a spectral detection means, and a line photosensor array 19 are arranged. The collimating lens 17 makes light emitted from the emission surface 16b of the fiber 16 light substantially parallel. The grating 18 disperses the parallel light from the collimating lens 17 in wavelength. The line photosensor array 19 receives and detects light wavelength-dispersed by the grating 18.

  The DMD 3, the CCD camera 13, and the line photosensor array 19 are electrically connected to a personal computer (hereinafter referred to as a PC) 20 as a measurement target area setting unit and a control unit, respectively. The PC 20 has functions such as setting and driving of the reflection pattern of the DMD 3, that is, ON / OFF pattern, accumulation of data acquired by the CCD camera 13 and the line photosensor array 19, readout, and arithmetic processing. In addition, driving and switching operations of each part of the microscope are performed as necessary.

  A monitor 21 is electrically connected to the PC 20 as display means. The monitor 21 displays the ON / OFF pattern of the DMD 3, the acquired image of the CCD camera 13, the acquired spectral information of the line photosensor array 19, and information necessary for calculation and electrical processing by the PC 20.

  Next, the operation of the embodiment configured as described above will be described.

  First, a projected image of the sample S will be briefly described. For example, when the objective lens 1 having a focal length of 9 mm and the imaging lens 9 having a focal length of 180 mm are used, the projection image of the specimen S formed by the objective lens 1 and the imaging lens 9, that is, the primary projection image is obtained by changing the specimen S to 20. The image is doubled (180 ÷ 9 = 20). In other words, the magnification of the objective lens 1 is 20 times, and the CCD camera 13 captures an enlarged image 20 times that of the sample S. On the other hand, for example, when the relay lens 14 having a focal length of 180 mm and the reduction lens 15 having a focal length of 9 mm are used, the relay image formed by the relay lens 14 and the reduction lens 15, i.e., the secondary projection image, has a primary projection image of 20 minutes. The image is reduced to 1 and the magnification from the sample S to the secondary projection image is 1.

  Accordingly, if the observation target range of the sample S is 0.2 mm in diameter, the secondary projection image projected onto the incident surface 16a of the fiber 16 has a diameter of 0.2 mm and receives light in the observation target range. For this purpose, a multimode fiber having a core diameter of 0.2 mm or more is used.

  In this way, the projection magnification of the secondary projection image and the diameter of the fiber 16 are appropriately determined according to the required observation target range of the sample S.

  Then, the secondary projection image formed on the incident surface 16a of the fiber 16 is transmitted by the fiber 16 as an integrated light amount of the secondary projection image, passes through the collimating lens 17, and is all taken into the grating 18. The light is detected and detected by the line photosensor array 19 after being wavelength-dispersed. That is, the integrated light quantity of fluorescence emitted from within the observation target region of the specimen S is acquired by being spectrally separated.

  Next, the procedure for measuring the spectral information of the sample S will be described in detail.

  In measuring the spectral information of the sample S, first, the ON / OFF pattern of the DMD 3 is set based on the image of the sample S obtained by the CCD camera 13. In this case, it is necessary to grasp in advance the relative error between the ON / OFF pattern of the DMD 3 and the projection image of the ON / OFF pattern of the DMD 3 acquired by the CCD camera 13 and to perform correction at the time of measurement.

  As a process for this, a “pre-measurement process” is executed according to the flowchart shown in FIG.

  First, in step 201, the fluorescent plate is placed at the position of the sample S, and the objective lens 1 is focused. In this case, it is desirable to use a glass plate or the like on which a fluorescent material is uniformly applied as the fluorescent plate.

  Next, in step 202, a reference pattern is created on the memory of the PC 20 and sent to the DMD 3. In this case, a reference pattern created in advance on the PC 20 is sent to the DMD 3 to form the reference pattern on the DMD 3. FIG. 4 shows an example of the reference pattern. In step 203, the fluorescent plate installed at the position of the sample S is excited with a reference pattern set on the DMD 3. The excitation light at this time is modulated by a reference pattern formed on the DMD 3 and then irradiated to the fluorescent plate. When excited by the reference pattern and emitted from the fluorescent plate, the fluorescence is acquired by the CCD camera 13 in the next step 204 and the projection image is stored in the memory on the PC 20.

  Next, in step 205, the reference pattern created in advance on the PC 20 and formed on the DMD 3 is compared with the projected image of the reference pattern acquired by the CCD camera 13, and the parallel movement amount, enlargement ratio, and rotation angle are calculated. In step 206, these values are stored in a memory (not shown) on the PC 20.

  Thereafter, when the ROI information designated on the PC 20 is sent to the DMD 3, a correction operation is performed based on these values, and the information is sent to the DMD 3. Thereby, at the time of measurement, the ROI designated on the PC 20 based on the image of the specimen S acquired by the CCD camera 13 and the ROI actually illuminated on the specimen S can be completely matched.

  The rotation angle can be corrected by rotating the CCD camera 13 itself. In this case, the correction of the rotation angle on the PC 20 can be omitted.

  Next, the spectral information of the sample S is measured.

  In this case, as a procedure for measuring the spectral information of the specimen S, a “measurement procedure” is executed according to the flowchart shown in FIG.

  First, in step 301, a projection image of the entire field of view of the specimen S is acquired by the CCD camera 13. In this case, the objective lens 1 is focused on the sample S including the measurement object. Next, the entire area of the DMD 3 is turned on so that the excitation light is uniformly applied to the sample S. In this state, the sample S is irradiated with the excitation light, and the fluorescence image is acquired by the CCD camera 13 ( FIG. 5 (A)).

  Next, in step 302, the acquired projection image is displayed on the monitor 21.

  Next, in step 303, a measurement object is selected from the display image of the monitor 21, and an ROI is set with a range including the measurement object as a measurement object region. In this case, as shown in FIG. 5B, while viewing the display image on the actual monitor 21, an arbitrary region emitting fluorescence is selected as a measurement object, and the ROI is set within a range including the measurement object. . The shape of the ROI at this time is generally a simple shape such as a circle or an ellipse as shown in the figure, but it can also be a complicated shape that draws the outline of the measurement object. is there.

  Next, in step 304, the information of each set ROI is stored in a memory (not shown) on the PC 20 individually. When all the ROIs are set, the entire area of the DMD 3 is turned off and the illumination on the specimen S is blocked.

  Next, in step 305, the direction of the observation light beam of the second observation optical path branching unit 12 is switched from the observation optical axis A3 to the observation optical axis A4. That is, the second observation optical path branching unit 12 is driven to switch the direction of the observation light beam from the observation optical axis A3 toward the CCD camera 13 to the observation optical axis A4 toward the line photosensor array 19.

  Next, in step 306, a single ROI is called from the information of each ROI stored in the memory of the PC 20 according to an appropriately determined order (hereinafter referred to as ROI # 1).

  Next, in step 307, for the # 1 ROI among the set ROIs, a correction operation is performed based on the calculation result obtained in the above-mentioned “pre-measurement processing”, and the first mask pattern as the reflection pattern is obtained. To construct.

  Next, in Step 308, the first mask pattern is sent to the DMD 3, and the ROI of # 1 on the specimen S is illuminated. In this case, the DMD 3 sets the inclination of the micromirror based on the first mask pattern. As a result, the excitation light is modulated and irradiated onto the specimen S. At this time, the irradiated area is only the area set as the ROI of # 1 (FIG. 5C).

  Next, in Step 309, the spectral information of the fluorescence of # 1 ROI is acquired by the line photosensor array 19, and the result is stored in the memory. In this case, the fluorescence emitted from the region set as # 1 ROI on the specimen S passes through the fiber 16, is split by the grating 18, and then enters the line photosensor array 19. Thereby, the fluorescence spectral information of the # 1 ROI is acquired, and this measurement result is stored in the memory of the PC 20.

  Thereafter, the same operation is repeated for all ROIs (step 310). That is, following # 1 ROI, for # 2 ROI, the fluorescence spectral information is acquired in the same procedure as described above, and the measurement result is stored in the memory of the PC 20 (FIG. 5 (D)). The same operation is performed for the # 3 ROI, the # 4 ROI..., And the same operation is repeated for all the ROIs, so that the fluorescence spectral information regarding all the set ROIs (FIG. 5E), It is stored in the memory of the PC 20.

  Accordingly, in this way, the ROI including the measurement object is set on the imaging screen of the specimen S, the reflection pattern of the DMD 3 is set by the mask pattern set based on the ROI, and the illumination light is applied to the specimen S. Since only the ROI including the measurement object can be selectively excited. Thereby, the spectral information of the fluorescence emitted from each ROI on the set specimen S can be measured with high accuracy without causing crosstalk due to unnecessary fluorescence from other regions.

Moreover, since the excitation light from the light source 5 is not irradiated to the ROI that is not the measurement target at each time point, it is possible to suppress the occurrence of unnecessary fading or phototoxicity change of the fluorescent material. .
Furthermore, since the accumulated light amount of the fluorescence emitted from the entire set ROI can be spectroscopically measured, it is possible to perform reliable spectroscopic measurement even when the density of the fluorescence emitted from the designated ROI is low.

  Further, since the spectral information of the measurement object at all positions in the observation field can be acquired with respect to the sample S, the sample S is moved, for example, the measurement object is moved to the center of the observation field during the measurement. This eliminates the need for efficient spectroscopic measurement.

  Furthermore, the spectral information can be acquired without scanning a two-dimensional region for one ROI, and the high-speed switching of the ROI by the DMD 3 can be performed, so that the time for spectroscopic measurement can be shortened.

(Second embodiment)
Next, a second embodiment of the present invention will be described.

  In this case, the schematic configuration of the multipoint fluorescence spectrophotometric microscope according to the second embodiment of the present invention is the same as that shown in FIG.

  This second embodiment is different from the “measurement procedure” in the same configuration as the first embodiment described above.

  In this case, the “measurement procedure” is executed according to the flowchart shown in FIG. 6 with the procedure for measuring the spectral information of the sample S.

  First, in step 601, a projection image of the entire field of view of the specimen S is acquired by the CCD camera 13. In this case as well, the objective lens 1 is focused on the sample S including the measurement object, and then the DMD 3 is turned on in the entire region so that the sample S is uniformly irradiated with the excitation light. S is irradiated with excitation light, and the fluorescence image is acquired by the CCD camera 13 (FIG. 7A).

  Next, in step 602, binarized image processing is performed to select the presence or absence of light emission from the acquired fluorescent image based on an appropriate threshold value. In this case, a threshold value is appropriately set based on the luminance value of the acquired fluorescent image, and binarized image data is acquired based on the presence or absence of light emission based on this threshold value.

  Next, in step 603, the contour of the measurement object is extracted from the binarized image data, and the ROI is automatically set. In this case, the binary boundary position is calculated from the binarized image data by the calculation processing of the PC 20, and the contour of the measurement object is extracted (FIG. 7B). Then, the area formed by this contour is automatically set individually as an ROI.

  In the above description, binarization is performed based on the luminance value of the fluorescent image, but in addition to this, binarization is performed based on the ratio of fluorescent images sequentially obtained using a plurality of different fluorescent filters. Also good.

  Next, in step 604, the information of each automatically set ROI is individually stored in the memory on the PC 20 (FIG. 7C). In addition, when the automatic setting of the ROI is completed, the entire area of the DMD 3 is turned off and the illumination on the specimen S is blocked.

  Next, in step 605, the direction of the observation light beam of the second observation optical path branching unit 12 is switched from the observation optical axis A3 to the observation optical axis A4. That is, the second observation optical path branching unit 12 is driven to switch the direction of the observation light beam from the observation optical axis A3 toward the CCD camera 13 to the observation optical axis A4 toward the line photosensor array 19.

  Next, in step 606, a single ROI is called from the information of each ROI stored in the memory of the PC 20 according to an appropriately determined order (hereinafter referred to as ROI # 1).

  Next, in step 607, the first mask pattern is constructed by performing a correction operation on the # 1 ROI among the automatically set ROIs based on the calculation result obtained in the above-mentioned “pre-measurement processing”.

  Next, in step 608, the # 1 mask pattern is sent to the DMD 3, and the # 1 ROI on the specimen S is illuminated. In this case, the DMD 3 sets the inclination of each micromirror based on the first mask pattern. As a result, the excitation light is modulated and irradiated onto the specimen S. At this time, the irradiated region is only the region set as the ROI of # 1 (FIG. 7D).

  Next, at step 609, the line photosensor array 19 acquires the spectral information of the # 1 ROI fluorescence and stores the result in memory. In this case, the fluorescence emitted from the region set as # 1 ROI on the specimen S passes through the fiber 16, is split by the grating 18, and then enters the line photosensor array 19. Thereby, the fluorescence spectral information of the # 1 ROI is acquired, and this measurement result is stored in the memory of the PC 20.

  Thereafter, the same operation is repeated for all ROIs (step 610). That is, following # 1 ROI, for # 2 ROI, fluorescence spectroscopic information is acquired in the same procedure as described above, and the measurement result is stored in the memory of the PC 20 (FIG. 7 (E)). The same operation is performed for the # 3 ROI, the # 4 ROI, and the like, and the same operation is repeated for all the ROIs, so that the fluorescence spectral information for all the set ROIs is stored in the memory of the PC 20. (FIG. 7F).

  Therefore, even in this case, the same effect as described in the first embodiment can be obtained. Furthermore, by performing binarized image processing for selecting the presence or absence of light emission with an appropriate threshold value for the luminance value of the fluorescent image of the sample S acquired by the CCD camera 13, a plurality of optimum ROIs for the sample S are obtained. It can be set automatically. Furthermore, by preparing as a processing program of the PC 20 a procedure for sequentially acquiring spectral information of a plurality of set ROIs, it becomes possible to automatically perform all the spectral measurements of the measurement object in the observation field of view, and thus the spectrophotometry The work for can be performed efficiently.

(Third embodiment)
Next, a third embodiment of the present invention will be described.

  FIG. 8 shows a schematic configuration of a multipoint fluorescence spectrophotometric microscope according to the third embodiment of the present invention, and the same parts as those in FIG.

  In this case, the third observation optical path branching unit 31 is disposed on the transmission optical path side viewed from the sample S of the mirror 2, that is, on the optical path between the absorption filter 8 and the imaging lens 9 on the extension of the observation optical axis A. ing.

  The third observation optical path branching section 31 enables switching of a plurality of prisms or insertion / removal of a single mirror (in FIG. 5, insertion / removal of a single mirror), and has advanced the observation optical axis A. The light is branched into two, an observation optical axis A5 that transmits light and an observation optical axis A6 that reflects light.

  An imaging lens 9 and a first observation optical path branching unit 10 are disposed on the observation optical path A5. The first observation optical path branching unit 10 enables switching of a plurality of prisms or insertion / removal of a single mirror (switching of a plurality of prisms in FIG. 5), and the light that has traveled along the observation optical axis A5. The observation optical axis A7 that is transmitted and the observation optical axis A8 that is reflected are branched into two.

  An eyepiece lens 11 is disposed on the observation optical axis A7. The eyepiece 11 is disposed at the imaging position of the imaging lens 9 so that the observation image of the sample S can be visually observed.

  On the observation optical axis A8, a CCD camera 13 as an imaging means is disposed. In the CCD camera 13, an imaging surface (not shown) is positioned at the focal position of the imaging lens 9 (a position optically conjugate with the focal position of the objective lens 1), and the projection image of the sample S is captured. .

  An imaging lens 32, a relay lens 33, a reduction lens 34, and an incident surface 16a of the optical fiber 16 as other imaging lenses are arranged on the observation optical axis A6 formed by reflecting the third observation optical path branching portion 31. ing. The relay lens 33 converts the observation light diverged after the projection image of the sample S is formed into a substantially parallel light at the focal position of the second imaging lens 32 (a position optically conjugate with the focal position of the objective lens 1). Is. The reduction lens 34 forms parallel light (projected image of the sample S) from the relay lens 33 with a reduction magnification. In the optical fiber 16, the incident surface 16 a is disposed at the focal position of the reduction lens 34, and a projection image of the sample S formed with a reduction magnification is incident thereon.

  Other configurations are the same as those in FIG.

  Next, the operation of the embodiment configured as described above will be described.

  First, a projected image of the sample S will be briefly described. For example, when the objective lens 1 having a focal length of 9 mm and the imaging lens 9 having a focal length of 180 mm are used, the projection image of the specimen S formed by the objective lens 1 and the imaging lens 9, that is, the primary projection image is obtained by changing the specimen S to 20. The image is doubled (180 ÷ 9 = 20). In other words, the magnification of the objective lens 1 is 20 times, and the CCD camera 13 captures an enlarged image 20 times that of the sample S. On the other hand, in the observation optical axis A6, for example, when a lens having a focal length of 90 mm is used as the second imaging lens 32, a projection image of the sample S formed by the objective lens 1 and the second imaging lens 32, that is, a primary projection image. Becomes an image obtained by enlarging the sample S 10 times at a position on the optical path 90 mm away from the second imaging lens 32 (90 ÷ 9 = 10). Further, when the relay lens 33 having a focal length of 90 mm and the reduction lens 34 having a focal length of 9 mm are used, the relay image formed by the relay lens 33 and the reduction lens 34, that is, the secondary projection image, is 1/10 of the primary projection image. The magnification from the sample S to the secondary projection image is 1.

  This magnification is the same as that described in the first embodiment, and in this case as well, it is appropriately determined according to the required observation target range of the sample S.

  Therefore, in this way, by using the imaging lens 32 independent from the imaging lens 9 used for visual observation of the microscope or observation with the CCD camera 13, without depending on the magnification of the microscope, An optical system for obtaining spectral information can be configured. Thus, by shortening the focal length of the second imaging lens 32 and the relay lens 33, the total length of the observation optical axis A6 from the third observation optical path branching section 31 to the fiber 16 can be shortened, and the compact configuration Can be realized.

  In addition, this invention is not limited to the said embodiment, In the implementation stage, it can change variously in the range which does not change the summary. For example, the magnification of the projected image of the specimen S described above is merely an example, and can be set as appropriate according to the size of the target measurement object and the required observation field of view. In addition, the objective lens 1 used in a normal microscope can selectively arrange a plurality of different magnifications on the observation optical axis. Similarly, in the present invention, a plurality of objective lenses 1 are used. Obviously, the magnification can be switched. Further, the observation optical axis reaching the CCD camera 13, the observation optical axis reaching the fiber 16, and the observation optical axis provided for visual observation, that is, what is arranged on the transmission side and the reflection side in each observation optical path branching portion. Is optional, and can be freely configured without departing from the gist of the present invention. Further, the illumination optical system can be configured not only in the epi-illumination but also in the transmission illumination. In the above description, an example of an inverted microscope in which the objective lens 1 is disposed on the lower side of the sample S has been described. However, the present invention can also be applied to an upright microscope in which the objective lens 1 is disposed on the upper side of the sample S.

  Furthermore, the above embodiments include inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent elements. For example, even if some constituent requirements are deleted from all the constituent requirements shown in the embodiment, the problem described in the column of the problem to be solved by the invention can be solved, and is described in the column of the effect of the invention. If the above effect is obtained, a configuration from which this configuration requirement is deleted can be extracted as an invention.

The figure which shows schematic structure of the multipoint fluorescence spectrophotometric microscope concerning the 1st Embodiment of this invention. The flowchart for demonstrating the pre-measurement process of 1st Embodiment. The flowchart for demonstrating the measurement procedure of 1st Embodiment. The figure which shows an example of the reference | standard pattern of 1st Embodiment. The figure explaining from acquisition of ROI of 1st Embodiment to acquisition of spectral information. The flowchart for demonstrating the measurement procedure of the 2nd Embodiment of this invention. The figure explaining from acquisition of ROI of 2nd Embodiment to acquisition of spectral information. The figure which shows schematic structure of the multipoint fluorescence spectrophotometric microscope concerning the 3rd Embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Objective lens, 2 ... Mirror 3 ... DMD, 4 ... Projection lens 5 ... Light source, 6 ... Collector lens 7 ... Excitation filter, 8 ... Absorption filter 9 ... Imaging lens, 10 ... Observation optical path branch part 11 ... Eyepiece lens, DESCRIPTION OF SYMBOLS 12 ... Observation optical path branch part 13 ... CCD camera, 14 ... Relay lens 15 ... Reduction lens, 16 ... Optical fiber 16a ... Incident surface, 16b ... Outgoing surface 17 ... Collimating lens, 18 ... Grating 19 ... Line photo sensor array, 20 ... PC
DESCRIPTION OF SYMBOLS 21 ... Monitor, 31 ... Observation optical path branch part 32 ... Imaging lens, 33 ... Relay lens, 34 ... Reduction lens

Claims (7)

A light source;
An illumination optical system for irradiating the sample with light from the light source;
An objective lens for forming the specimen image;
A micro deflection element provided at a position optically conjugate with the sample in the illumination optical system;
Imaging means for imaging a specimen image formed by the objective lens;
A measurement target area setting means for setting a measurement target area on the sample from a captured image of the imaging means;
Control means for controlling the light deflection pattern of the micro deflection element in accordance with the measurement target area set by the measurement target area setting means;
Spectral detection means for obtaining wavelength-dispersed light generated from the measurement target region on the specimen;
A multi-point fluorescence spectrophotometric microscope characterized by comprising: A light source;
An illumination optical system for generating light from the light source;
An objective lens for condensing the light from the illumination optical system on the specimen;
A micro deflection element provided at a position optically conjugate with the focal position of the objective lens in the illumination optical system;
Imaging means for imaging light generated from the specimen;
A measurement target area setting means for setting a measurement target area on the sample from a captured image of the imaging means;
Control means for controlling the light deflection pattern of the micro deflection element in accordance with the measurement target area set by the measurement target area setting means;
Spectral detection means for obtaining wavelength-dispersed light generated from the measurement target region on the specimen;
A multi-point fluorescence spectrophotometric microscope characterized by comprising: A light source;
An illumination optical system for generating light from the light source;
An objective lens for condensing the light from the illumination optical system on the specimen;
A micro deflection element provided at a position optically conjugate with the focal position of the objective lens in the illumination optical system;
An imaging unit that is disposed at a position optically conjugate with the focal position of the objective lens in the first observation optical system having an imaging lens that passes through the objective lens;
A fiber for transmitting the projection image of the specimen, the incident surface being arranged at a position optically conjugate with the imaging means;
Spectral detection means for obtaining wavelength-dispersed light emitted from the fiber;
A measurement target area setting means for setting a measurement target area on the sample from an imaging screen of the imaging means;
Control means for controlling the deflection pattern of the micro deflection element according to the measurement target area set by the measurement target area setting means, and processing the acquired data of the spectral detection means;
A multi-point fluorescence spectrophotometric microscope characterized by comprising: The multipoint fluorescence spectrophotometric microscope according to any one of claims 1 to 3, wherein the measurement target region setting unit sets an arbitrary region emitting light from the imaging screen as a measurement target region. The measurement target area setting unit sets the measurement target area based on binarized data of a luminance value of light emitted from the imaging screen. Multipoint fluorescence spectrophotometric microscope. The incident surface of the fiber is disposed at a position optically conjugate with the imaging means in the second observation optical system having another imaging lens that passes through the objective lens. 5. The multipoint fluorescence spectrophotometric microscope according to any one of 5 above. A first step of acquiring a captured image of the entire field of view of the specimen irradiated with light from the light source via the illumination optical system;
A second step of setting a measurement target region on the specimen from the captured image acquired in the first step;
A third step of controlling a deflection pattern of a micro deflection element provided at a position optically conjugate with the sample of the illumination optical system in accordance with the measurement target region set in the second step;
And a fourth step of acquiring the wavelength generated light from the measurement target region on the specimen irradiated with light through the micro deflection element whose deflection pattern is controlled in the third step. A multipoint fluorescence spectrophotometric method characterized by the above.

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