JP2002014043A - Multicolor analyzer for microscope, and multicolor analysis method using microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an apparatus and a method where fluorescence and Raman scattering light are detected easily and satisfactorily, and the advantage of fluorescence spectroscopy and Raman spectroscopy can be fully utilized. SOLUTION: The analyzer is provided with a laser light source 2, and a three-dimensional movement base which moves and operates a sample S. Excitation light generated when the sample S is irradiated with a laser beam, is detected by an image pickup element 3 via a microscope 1 and a spectroscope 31 so as to be image-processed by an image processing means.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a multi-color analysis for a microscope, in which a large number of scattered lights generated over a wide wavelength range when a sample is irradiated with a laser beam for excitation are simultaneously detected by a CCD detector and displayed as an image. The present invention relates to an apparatus and a multicolor analysis method using a microscope that detects each scattered light obtained from the sample and displays it as a combined image or a single image for each spectrum.

[0002]

2. Description of the Related Art Conventionally, there has been proposed an apparatus for detecting, via a microscope, excitation light, fluorescence, Raman scattered light, and the like generated when a sample is irradiated with a laser beam for excitation. By detecting Raman scattered light or the like emitted from a sample using such an apparatus, the chemical structure and physical state of a substance contained in the sample can be specified. Further, according to such an apparatus, nondestructive detection can be performed regardless of whether the sample is a solid, liquid, or gas.

In such an apparatus, in order to detect fluorescence, Raman scattered light, and the like, an interference filter (bandpass filter), a liquid crystal tunable filter (LCTF),
An optical element such as an acousto-optic filter (AOTF) or an interferometer Fourier transform spectroscopy (TF) is used.
Since these optical elements have wavelength selectivity, they can detect only Raman scattered light having a specific wavelength emitted from a sample.

[0004]

The interference filter (bandpass filter), which is an optical element for wavelength selection, used in the above-described apparatus has a wavelength wavelength resolution of about 5 nm and a transmittance. Is 30% or more. This optical element is inexpensive and can cover a wide wavelength band from ultraviolet to near infrared, but it can only detect fluorescence, and it is difficult to replace a filter for each wavelength band.

Further, a liquid crystal tunable filter (LCT)
In F), the wavelength resolution is about 5 nm to 10 nm, and the transmittance is 15% or less. This optical element can perform detection with polarized light and high-speed wavelength sweep in the visible light band, but can detect only fluorescent light, has a narrow wavelength band that can be covered, and has a fixed wavelength resolution.

The acousto-optic filter (AOTF) has a wavelength resolution of about 1 nm to 10 nm and a transmittance of about 40%. This optical element can perform detection with polarized light and high-speed wavelength sweep in the visible light band, but can detect only fluorescent light, has a narrow wavelength band that can be covered, and has a fixed wavelength resolution.

Next, in the interferometer Fourier transform spectroscopy (TF), the wavelength resolution is about 10 nm, but the wavelength width on the long wavelength band side is 5 nm or less at 400 nm and 20 nm or less at 780 nm. Become wider.
This optical element has a high transmittance of about 90% and can cover from the visible light band to the near-infrared ray, but cannot detect by polarization, can detect only fluorescence, and can be calculated by FFT for 10 to 50 seconds. And slow is the drawback. Further, it is difficult to exchange elements for each wavelength band and to have different wavelength resolutions for each wavelength band.

Conventionally, it has not been possible to perform a rapid measurement, for example, it takes several hours to measure a spectrum having a plurality of peaks for each peak (multicolor), for example, for four colors.

As described above, in the conventional optical element,
There was no device capable of detecting fluorescence and Raman scattered light satisfactorily, and there was no device capable of fully utilizing the advantages such as Raman spectroscopy.

Therefore, the present invention has been proposed in view of the above-mentioned circumstances, and is intended to easily and satisfactorily detect Raman scattered light and to fully utilize the advantages of Raman spectroscopy. An object of the present invention is to provide a multicolor analyzer for microscopes and a multicolor analysis method using a microscope.

[0011]

In order to solve the above-mentioned problems, a multicolor analyzer for a microscope according to the present invention is provided with a laser light source for irradiating a sample with a laser beam for excitation, and supporting and moving the sample. A three-dimensional moving table, a microscope into which a large number of scattered lights over a wide wavelength range emitted from a sample by being irradiated with an excitation laser beam, a spectroscope for dispersing the scattered light passing through the microscope, and a spectroscope An image sensor for receiving scattered light passing through the imager, an image processing means for processing an output signal from the image sensor and outputting the image signal as an image signal;
And a control means for controlling the three-dimensional moving table and the image processing means.

In the multicolor analyzer for a microscope, the image processing means simultaneously measures a plurality of spectra for each point on the sample in accordance with the movement of the sample by the three-dimensional moving table. Is what you do.

Further, in this multicolor analyzer for microscopes, the image processing means displays an area on the sample where scattered light has a specific wavelength.

In the multicolor analysis method using a microscope according to the present invention, the sample is irradiated with a laser beam for excitation, the sample is moved, and the sample is irradiated with the laser beam for excitation. A large number of scattered lights over a range are received by an image sensor via a microscope and a spectroscope, and an output signal from the image sensor is processed and output as an image signal using an image processing circuit controlled by a computer device. It is assumed that.

In the multicolor analysis method using the microscope, a plurality of spectra are simultaneously measured for each point on the sample by the image processing means according to the movement of the sample. It is.

Further, the multicolor analysis method using the microscope is characterized in that an image processing means displays an area on the sample where the scattered light has a specific wavelength.

[0017]

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

The multicolor analysis method using a microscope according to the present invention is carried out by a multicolor analyzer for a microscope according to the present invention described below. As shown in FIG. 1, the microscope multicolor analyzer according to the present invention irradiates a sample S installed in an objective lens block 1 with an excitation laser beam emitted from a laser light source 2. Components such as Raman scattered light and fluorescence generated from the sample S by irradiation are illuminated through a confocal microscope and a spectroscope into a solid-state image sensor (charge-coupled device (CCD: ch:
arge-coupled device)) 3.

As shown in FIG. 2, the sample S is supported and moved by a piezoelectric stage 33 serving as a three-dimensional moving table. The piezoelectric stage 33 is configured to support a mounting table via a piezo element. The piezoelectric stage 33 operates under the control of a computer device 34 serving as control means via a piezoelectric stage controller circuit 32a. The sample S supported on the mounting table is moved according to the execution of the software.

As shown in FIG. 1, the overall configuration of this multicolor analyzer for a microscope is as follows. A light beam emitted from a laser light source 2 passes through mirrors 13 and 14 and firstly a condenser lens 15 and a pinhole. After passing through the mask 16 and the collimator lens 17, the light is converted into a parallel light beam. This parallel light flux further passes through mirrors 18 and 11 and is in a circularly polarized state.
The light is incident on the beam splitter 8. The light beam incident on the beam splitter 8 passes through the first condensing lens 4 constituting the confocal microscope and the pinhole 6 of the pinhole mask 5, and passes through the mirror 19 in the objective lens block unit 1. The light is incident on the second condenser lens 7 and is focused on the sample S. Here, the second condenser lens 7 includes a collimator lens 7a and an objective lens 7b.

The light beam condensed on the sample S is reflected by the sample S, including scattered light, and converges via the second condensing lens 7, while again converging on the pin of the pinhole mask 5. Returning to the hole 6, an image is formed in the pinhole 6. The reflected light flux that passes through and diffuses in the pinhole 6 is returned to a parallel light flux by the first condenser lens 4,
The process returns to the beam splitter 8, which is a light beam branching unit that guides the reflected light beam to the detection unit.

In the beam splitter 8, the sample S
The reflected light flux from the light source is 90% of light in a band other than a narrow wavelength band of about 2 nm centered on the oscillation wavelength of the laser light source 2, that is, a light flux containing components due to Raman scattering or fluorescence.
The light is transmitted at a high transmittance and is branched from the optical path returning to the laser light source side. The luminous flux transmitted through the beam splitter 8 is
Further, the light passes through notch filters (Notch-filters) 12 and 20, and the wavelength is selected. Note that this notch filter 1
The reflected light flux is perpendicularly incident on 2,20. The luminous flux having passed through the notch filters 12 and 20 is introduced into a spectral block 31 forming a spectroscope via a mirror 21, an interference filter 27, and a third condenser lens 9.

In the dispersing block 31, the reflected light beam is reflected by mirrors 22 and 23, passes through a reflection type diffraction grating (grating) 24, and is further reflected by mirrors 25 and 30.
And is detected by the solid-state imaging device 3. The reflection type diffraction grating 24 can be replaced with a different one by a so-called turret type.

As described above, the luminous flux incident on the solid-state imaging device 3 via the optical system in the spectral block 31 is
The solid-state imaging device 3 can simultaneously measure the entire spectral region dispersed in the spectral block 31. When the reflected light beam is observed by the solid-state imaging device 3, light measurement in a short time is possible. Further, it is possible to perform exposure for a long time such as tens of minutes.

As shown in FIG. 3, the solid-state image pickup device 3 receives the light flux split through each optical element constituting the light splitting block 31. At this time, as shown in FIG. 4, the solid-state imaging device 3 receives, for example, a spectrum including a plurality of peaks in a range of 300 nm to 700 nm in 1024 steps. Note that the wavelength sweep in the spectral block 31 is not performed. Further, no slit is provided between the spectral block 31 and the solid-state imaging device 3, and the spectral block 31 is not provided.
Is received by the solid-state imaging device 3.

In this multi-color analyzer for microscopes, as shown in FIG. 2, the imaging device 3 operates under the control of the computer device 34 via the imaging device controller circuit 32b. The computer device 34 also controls the spectral block 31. In addition,
As shown in FIG. 1, the piezoelectric stage controller circuit 32a and the imaging element controller circuit 32b are disposed near the spectral block 31 as the controller 32.

Further, the computer device 34 includes an image processing circuit serving as image processing means for processing an output signal from the solid-state imaging device 3 and outputting the processed image signal as an image signal. As a control means for controlling the image processing circuit, Also works.

In the multicolor analyzer for a microscope, a spectrum can be measured for each point on the sample S according to the movement of the sample S by the piezoelectric stage 33 under the control of the computer device 34. . That is, in this multicolor analyzer for a microscope, as shown in FIG. 5, the sample S on the piezoelectric stage 33 is irradiated with a laser beam for excitation, and the position of the laser beam is fixed. , Piezoelectric stage 33
Is measured in a three-dimensional direction indicated by arrows X, Y, and Z in FIG. 5 to measure the spectrum at each point on the sample S as shown in FIG. is there.

In this multicolor analyzer for microscopes, multicolor analysis of 8 to 20 colors can be performed in ten and several seconds, which is extremely useful, for example, in DNA analysis.

In this multicolor analyzer for microscopes, by changing the magnification of the confocal microscope, a part of the sample S is enlarged as shown in FIG. Can also be measured. How many points of the spectrum are measured on the sample S can be set by selecting the step width of scanning by the piezoelectric stage 33 according to the size of the sample S and the magnification of the confocal microscope. .

In the computer device 34, as shown in FIG. 7, spectrum data for each point on the sample S is obtained, and this data is processed to determine the distribution status and intensity of the scattered light wavelength. It can be displayed as a two-dimensional image or a three-dimensional image.

Further, the computer device 34 controls the sample S
By processing the spectrum data obtained from each of the above points, it is possible to display only the region where the scattered light has a specific wavelength on the sample S as shown in FIG. In FIG. 8, the lower graph shows a spectrum including a plurality of peaks, and the image drawn on the upper graph shows
The image which shows the area | region which emits the scattered light of the wavelength band selected corresponding to each peak is shown.

In this multicolor analyzer for a microscope, a high wavelength resolution of about 0.1 nm can be obtained.
The transmittance is as high as about 40% to 90%. It can cover a wide band from the ultraviolet band to the near infrared band, can detect by polarization, and can measure not only fluorescence but also Raman scattered light. Further, an operation such as replacement of an optical element is not required from the ultraviolet band to the near infrared band.

In order to execute the multicolor analysis method using the microscope according to the present invention in the multicolor analyzer for microscope according to the present invention configured as described above, FIG.
First, in step S1, setting of initial conditions, setting of a scanning area by the piezoelectric stage 33, setting of a step width of scanning, setting of wavelength resolution, and the like are performed in step S1. Further, the reflection type diffraction grating 24, the filter, and the slit in the spectral block 31 are controlled, and the data acquisition time, temperature, various modes, and the like of the solid-state imaging device 3 are set.

Next, in step S2, a spectrum is measured.

Further, in step S3, the number of selected wavelength bands and the wavelength width in each selected wavelength band are set.

In step S4, by repeating the movement of the sample S by the piezoelectric stage 33 and the measurement of the spectrum, spectrum data is obtained for each point on the sample S, and the three-dimensional spectrum of the sample S is obtained. Perform data mapping.

In step S5, image processing is performed based on the spectrum data obtained up to step S4. At this time, the computer device 34 can display the distribution status and intensity of the scattered light wavelength as a two-dimensional image or a three-dimensional image. Image processing of displaying can also be performed.

[0039]

As described above, in the multicolor analyzing apparatus for a microscope and the multicolor analyzing method using the microscope according to the present invention, a high wavelength resolution of about 0.1 nm can be obtained, and the transmittance is 40% to 40%. It is as high as about 90%. It can cover a wide band from the ultraviolet band to the near infrared band, can detect by polarization, and can measure not only fluorescence but also Raman scattered light. Further, an operation such as replacement of an optical element is not required from the ultraviolet band to the near infrared band.

In the multicolor analyzer for microscope and the multicolor analysis method using the microscope according to the present invention, multicolor analysis of 8 to 20 colors can be carried out in ten and several seconds. It is extremely useful in analysis and the like.

That is, the present invention provides a multicolor analyzer for a microscope and a microscope which can easily and satisfactorily detect fluorescence and Raman scattered light and can fully utilize the advantages of fluorescence and Raman spectroscopy. The present invention can provide a multicolor analysis method used.

[Brief description of the drawings]

FIG. 1 is a plan view showing a configuration of a multicolor analyzer for a microscope according to the present invention.

FIG. 2 is a block diagram showing a configuration of the multicolor analyzer for a microscope.

FIG. 3 is a side view showing a relationship between a spectral block and a solid-state imaging device in the multicolor analyzer for a microscope.

FIG. 4 is a front view showing a spectrum (multicolor) having two or more peaks received by the solid-state imaging device.

FIG. 5 is a perspective view showing a sample supported on a piezoelectric stage in the microscope multicolor analyzer.

FIG. 6 is a plan view showing the relationship between a sample and spectrum measurement points in the multicolor analyzer for a microscope.

FIG. 7 is a perspective view showing a relationship between spectrum measurement points and obtained spectrum data in the multicolor analyzer for a microscope.

FIG. 8 is a graph showing a relationship between peaks of many spectra measured by the multicolor analyzer for a microscope and an image obtained by using each peak as a selected wavelength band.

FIG. 9 is a flowchart illustrating an operation when the multicolor analysis method for a microscope using the microscope according to the present invention is executed in the multicolor analysis apparatus for a microscope.

[Explanation of symbols]

1 objective lens block section, 2 laser light source, 3 solid-state imaging device, 31 spectral block, 32 controller,
33 piezoelectric stage, 34 computer device

Continued on the front page F term (reference) 2G043 EA01 EA03 FA02 GA02 GA07 GB03 GB19 HA07 HA09 JA02 JA04 KA01 KA02 KA03 KA07 KA09 LA03 NA01 2H052 AA08 AC04 AC15 AC34 AD20 AD34 AF07 AF14 AF25

Claims (6)

[Claims] 1. A laser light source that irradiates a sample with a laser beam for excitation, a three-dimensional moving table that supports and moves the sample, and a wide wavelength range that the sample emits when irradiated with the laser beam for excitation. A plurality of scattered light incident thereon, a spectroscope for dispersing the scattered light passing through the microscope, an imaging device for receiving the scattered light passing through the spectroscope, and processing an output signal from the imaging device. A multi-color analyzer for a microscope, comprising: image processing means for outputting the image signal as an image signal; and control means for controlling the three-dimensional moving table and the image processing means. 2. The multi-purpose microscope according to claim 1, wherein the image processing means simultaneously measures a plurality of spectra for each point on the sample according to the movement of the sample by the three-dimensional moving table. Color analyzer. 3. The multicolor analyzer for a microscope according to claim 1, wherein the image processing means displays an area where the scattered light has a plurality of specific wavelengths on the sample. 4. A sample and a spectroscope that irradiate a sample with an excitation laser beam, move the sample, and irradiate the sample with the excitation laser beam to emit a large number of scattered lights over a wide wavelength range emitted by the sample. A multi-color analysis method using a microscope, wherein light is received by an image sensor through the device and an output signal from the image sensor is processed and output as an image signal using an image processing circuit controlled by a computer device. . 5. A multicolor analysis method using a microscope according to claim 4, wherein a plurality of spectra are simultaneously measured for each point on the sample by the image processing means in accordance with the movement of the sample. . 6. A multicolor analysis method using a microscope according to claim 4, wherein the image processing means displays an area where the scattered light has a plurality of specific wavelengths on the sample.