JPH0372238A - Highly sensitive microscopic multi-wavelength spectroscope - Google Patents

Abstract

PURPOSE:To enable spectral analysis of very weak emission with a microscopic multi-wavelength by providing an optical system to emit light from a point or a line source which is formed from a microscopic area of a magnified image of a sample or from an area with a certain extent. CONSTITUTION:An optical system 3 which emits light from a microscopic area of a magnified image Sb of a sample S or from an area with a certain extent being limited to a spot or a line makes the light incident on a collimator lens 4 which makes light from the magnified image Sb of the sample S incident on a reflection type diffraction grating 5 being turned into a parallel light beam with a higher parallelism without leaking. Therefore, the diffraction grating 5 delivers sufficient resolutions while diffracting a very weak light. Then, an imaging lens 6 forms an image of the parallel light diffracted by the diffraction grating 5 on an image surface P as spectral image. Then, a spectral analysis is performed with a highly sensitive one-dimensional or two-dimensional detector 7 disposed on the spectral image surface based on the control of a computer 8.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention is a method for analyzing the spectrum of extremely weak light ranging from the visible region to the infrared, such as bioluminescence, chemiluminescence, and extremely weak fluorescence caused by excitation light, by analyzing the image size using a microscope. This invention relates to a high-sensitivity microscopic multi-wavelength spectroscopy device that is bright, compact, and has excellent operability.

[Conventional technology]

Microscopic multi-wavelength spectrometers have traditionally been used to measure the absorption spectral characteristics of microscopic samples and thereby quantitatively or qualitatively investigate absorbing substances contained in the samples. Recently, bioluminescence, chemiluminescence,
It has also been developed as a device for measuring the spectral characteristics of extremely weak luminescence from minute objects that exhibit extremely weak fluorescence due to excitation light.

Among these methods, microspectrophotometry using a spectrometer using a diffraction grating and Fourier transform for microscopic measurement using a Fourier transform spectrometer are used to determine the absorption spectral characteristics and reflection spectral temporality of excitation light by a sample. Spectroscopy has been put into practical use. These spectroscopic characteristics can be determined by detecting an intensity several orders of magnitude lower than the intensity for each wavelength of the excitation light, and even difficult samples can be measured by increasing the excitation light or diluting the sample concentration. It does not require the highest sensitivity measurement system that can detect ultra-weak light with ultra-high sensitivity.

On the other hand, measurements of fluorescence and Raman scattering from a sample using excitation light under a microscope include microfluorescence spectroscopy for measuring the concentration of intracellular calcium, microfluorescence spectroscopy using Fourier transform spectroscopy using an interferometer, and It has been put into practical use as micro-Raman spectroscopy under a microscope. However, fluorescence and Raman scattering detect much weaker light than measuring transmitted light in order to determine the absorption spectral characteristics of a sample, so microscopic spectroscopy as bright as possible is required.

Furthermore, the brightest possible microscopic spectroscopy is required for spectroscopy of extremely weak biophotons emitted from living organisms and emitted light without excitation light, such as chemiluminescence. That is, in microscopic fluorescence spectroscopy, microscopic Raman spectroscopy, and microscopic luminescence spectroscopy, the light emission from a minute portion is magnified using a microscope and the amount of light decreases in inverse proportion to the magnification. Therefore, as the magnification increases, microscopic spectroscopy becomes increasingly difficult. Therefore, a highly sensitive microspectroscopy device is required.

However, in order to perform spectroscopy of such extremely weak light under a microscope, it is desirable to perform measurements with as little light loss as possible. For this purpose, it is preferable to use an optical system that can have a large output solid angle in order to increase the luminous flux utilization rate (throughput), and a detection system that has the advantage of simultaneous photometry that measures light of many wavelengths at once. In other words, a highly sensitive multi-wavelength spectrometer is required that uses bright optical systems for both the collimator system and the condensing system, and also incorporates the design concept of a polychromator that allows spectrum analysis of multiple wavelengths simultaneously without wavelength scanning. . However, none of the conventional spectroscopic devices satisfies this requirement.

In other words, since conventional spectrometers using diffraction gratings are basically monochromators, they require an entrance slit and an exit slit, which necessitates wavelength scanning, in which case light other than the exit slit is discarded. Because it is closed, there is no advantage to simultaneous photometry. In addition, since the configuration does not include a one-dimensional or two-dimensional light distribution detector that detects the spectral distribution on the exit surface, simply removing the exit slit and attaching a detector will cause focal plane problems and aberrations. There's a problem and it's not working. In addition, since both the collimator system and the condensing system use reflecting mirrors off-axis, aberrations cause bending of the spectral line image, making it impossible to reduce the F number very much, and there is a limit to the brightness of the spectrometer. Those with F=3 or less have not been put into practical use. Recently, an F=1 spectrometer has been developed using a parabolic mirror as a reflecting mirror in such a conventional spectrometer. However, since the condensing mirror (parabolic mirror) is used off-axis, the spectral line image is curved, and it is difficult to obtain accurate spectral distribution by arranging a one-dimensional or two-dimensional light distribution detector. In addition to the major problem that it is difficult to use a detector with high sensitivity, it is necessary to cool the detection surface.Therefore, as a means to prevent dew condensation on the detection surface, photoelectric conversion of one-dimensional or two-dimensional light distribution detectors is There is a problem in that the focal length of the condensing parabolic mirror must be increased because a relatively thick insulating vacuum chamber must be placed in front of the surface. Furthermore, polychromators that use a concave diffraction grating and an array detector as a detector have recently been sold, but there are limits to the diameter and radius of curvature of the concave diffraction grating.
The brightness is limited and insufficient. On the other hand, for the spectroscopic analysis of extremely weak luminescence, it has been proposed to use stationary interferometry using a three-sided common-path interferometer, a square common-path interferometer, a birefringent polarization interferometer, etc., but further studies are needed. As a result, questions have been raised about the claim that it is brighter than a spectrometer because the area of the luminescent sample surface is larger. Furthermore, these interferometers employ optical systems that are brighter than conventional spectrometers, but this is due to the use of brighter lenses instead of mirrors, and is not a characteristic of the interferometer.

Currently, the width of the detector array is smaller than the width of the diffraction grating, and the size of these widths determines the resolution of each spectroscopic system, so the resolution of stationary interferometry is higher than that of the spectrometer. Moreover, from the point of view of energy, in the case of a spectrometer, increasing the width of the entrance slit increases energy at the cost of deteriorating the resolution by 1, but this cannot be done with stationary interferometry.

For example, the 11th model is a system that combines a conventional laser Raman spectrometer and a microscope to be able to measure Raman spectra in a local area of about 1 μm.
Although a microscopic Raman system having the block diagram shown in the figure has been developed, this microscopic Raman system has a complicated coupling mechanism between the microscope section and the laser Raman spectrometer section.

[Problem to be solved by the invention]

Due to the current situation as described above, spectroscopic analysis of extremely weak luminescence from microscopic objects exhibiting bioluminescence, chemiluminescence, extremely weak fluorescence due to excitation light, etc. using conventional spectrometers or spectroscopy, especially at multiple wavelengths at the same time. It was difficult to determine the spectral distribution of

Therefore, it is an object of the present invention to overcome the drawbacks of the conventional spectrometers or microspectroscopy devices using spectroscopy, and to provide an extremely bright and compact device capable of emitting extremely weak fluorescence such as bioluminescence, chemiluminescence, and extremely weak fluorescence caused by excitation light. An object of the present invention is to provide a high-sensitivity microscopic multi-wavelength spectrometer using a novel high-sensitivity multi-wavelength spectrometer based on the technical idea of a polychromator, which can simultaneously determine the spectral distribution of weak light without wavelength scanning.

[Means to solve the problem]

The high-sensitivity microscopic multi-wavelength spectrometer of the present invention includes a microscope optical system that forms an enlarged image of a sample, and a system that confines light from a minute region or a region with a certain degree of spread in the enlarged image of the sample to a point or line. A bright collimator lens that focuses the point or linear exit end of this limited output optical system and takes in the light emitted from it and makes it parallel, and the light that is made parallel by the collimator lens. a reflection-type diffraction grating that diffracts and separates the light, an imaging lens that images the parallel light separated by the reflection-type diffraction grating as a spectral image on an image plane, and a It is characterized in that it is comprised of a highly sensitive multi-wavelength spectrometer consisting of a highly sensitive one-dimensional or two-dimensional photodetector.

For spectroscopy of ultra-weak luminescence caused by excitation light, the microscope optical system has a light source for excitation of the sample that can fix the wavelength or change the wavelength, and the sample is irradiated by the excitation light source. It is configured to form an enlarged image of re-emitted light from the sample.

For spectroscopic detection of extremely weak natural luminescence such as bioluminescence and chemiluminescence, a highly sensitive multi-wavelength spectrometer uses a blazed diffraction grating as a reflection type diffraction grating, and each component of the spectrometer is It is desirable to arrange the diffracted light so that it can be taken out as a spectral image.For spectroscopic detection of extremely weak light emitted by excitation light such as Raman scattering and fluorescence, a blazed diffraction grating is used as a reflection diffraction grating. It is desirable to arrange each component so that the +1st-order diffracted light can be taken out as a spectral image.

As a modification of the above-mentioned high-sensitivity multi-wavelength spectrometer, one dual-purpose lens is used as both the collimator lens and the imaging lens, and this dual-purpose lens is arranged parallel to the front surface of the reflection type diffraction grating, so that the focal plane of the dual-purpose lens is The dot or line exit end of the optical system that confines light from a minute area of the magnified image of the sample or an area with a certain degree of spread to a dot or line and outputs it is positioned above the dual-purpose lens. It is also possible to arrange a highly sensitive one-dimensional or two-dimensional photodetector on the image plane.

In addition, as an optical system that confines light from a microscopic region of an enlarged image of the sample or a region with a certain degree of spread to a point or line and emits it, a slit or a pinhole may be used, or a light concentrator may be used. You may also use

Furthermore, the high sensitivity 1 of the high-sensitivity multi-wavelength spectrometer as described above
By arranging two or more spectrometers of the same type or different types in multiple stages, excluding a dimensional or two-dimensional photodetector, and arranging them for additive dispersion or differential dispersion, angular dispersion can be achieved by utilizing the diffracted light several times. By controlling this, a highly sensitive multi-wavelength spectrometer with improved resolution can be obtained.

[Effect]

In the high-sensitivity multi-wavelength spectrometer used in the high-sensitivity multi-wavelength microspectroscopy device of the present invention, light from a minute region of an enlarged image of a sample or a region with a certain degree of spread is limited to a point or line. The optical system that emits the light from the magnified image of the sample formed by the microscope optical system enters the collimator lens as a point or linear light source, and the collimator lens adjusts the parallelism without leaking the light from the magnified image of the sample. The parallel light beam is made into a highly parallel light beam and is made incident on a reflection type diffraction grating. therefore,
Reflection-type diffraction gratings fully demonstrate their resolution and are capable of dispersing extremely weak light. Furthermore, since the collimator lens and the imaging lens can be arranged very close to the reflective diffraction grating, the device can be made smaller. And, as the collimator lens and imaging lens, bright lenses with the smallest possible F number can be used.
The brightness of the entire high-sensitivity multi-wavelength spectrometer, which is a composite thread of both lenses, can be sufficiently increased, and by combining it with a high-sensitivity one-dimensional or two-dimensional photodetector, bioluminescence,
Simultaneous multi-wavelength analysis of ultra-weak light such as chemiluminescence and ultra-weak fluorescence caused by excitation light is now possible, especially bioluminescence, chemiluminescence,
It is effective as a means of researching living organisms and trace components using extremely weak fluorescence caused by excitation light. Therefore, the high-sensitivity microscopic multi-wavelength spectrometer of the present invention is a multi-wavelength spectrometer that simultaneously captures multiple wavelengths of ultra-weak light from minute objects exhibiting bioluminescence, chemiluminescence, ultra-weak fluorescence due to excitation light, etc. as described above. Since it uses a highly sensitive multi-wavelength spectrometer that can easily perform spectroscopic analysis, it is bright, can detect ultra-weak light, is small and has excellent operability, and is particularly useful for bioluminescence, chemiluminescence, and excitation light. This will be an effective means of studying living organisms using extremely weak fluorescence.

〔Example〕

The high-sensitivity microscopic multi-wavelength spectrometer of the present invention combines a microscope optical system and a multi-wavelength spectroscopic device, and the microscope optical system itself consists of an objective lens, an eyepiece lens, or an imaging lens, as in a conventional microspectroscopic device. Various types of microscope optical systems can be used. However, it uses a spectroscopic device that is brighter and more sensitive than conventional ones, and is also used as a multi-wavelength simultaneous detection device.

In other words, in order to perform spectroscopic analysis of extremely weak light such as bioluminescence, chemiluminescence, and extremely weak fluorescence caused by excitation light using a multiwavelength spectrometer, it is necessary to analyze the light from the luminescent point of the sample magnified by the microscope optical system. It is necessary to select an optical system so that as much light as possible enters the spectroscopic system. Therefore, it is necessary to make the solid angle at which the optical system looks at the extremely weak light emitting point as large as possible.

On the other hand, the spectral line image separated by the spectroscopic system must be formed as linearly as possible.

At present, the only optical system that satisfies these requirements is an optical lens with a small F number that is made up of a combination of optical glass lenses. Optical lenses with no aberrations and an F number of l or less can be easily produced by hand. Therefore, in the highly sensitive multi-wavelength spectrometer used in the present invention, such an optical lens with a small F number is used.

Then, a reflection type diffraction grating is used as a spectroscopic system that separates the light beam made parallel by this optical lens, and further,
A bright optical lens is used to image the diffracted light and provide a spectral distribution image, and a highly sensitive one-dimensional or two-dimensional photodetector is placed on the image plane to electrically detect the spectral intensity distribution image. The light intensity of each wavelength is detected simultaneously.

FIG. 1 shows an example of the overall configuration of a high-sensitivity microscopic multi-wavelength spectrometer according to the present invention. The high-sensitivity microscopic multi-wavelength spectrometer shown in this drawing consists of a microscope optical system r that forms an enlarged image of the sample S, and a spectroscopic detection optical system (high-sensitivity The microscope optical system I is a multi-wavelength spectrometer).
an illumination light source (not shown; not necessary if the sample itself generates extremely weak light), a sample mounting table (not shown), an objective lens system 1 having an image-side focal point Fl, and an image-side focal point. F2
It consists of an eyepiece lens system 2 having a. In this microscope optical system (2), an intermediate image Sa of the sample S is formed by the objective lens system 1 at a distance of 11 from the image-side focal point Fl of the objective lens system, and an enlarged image sb of the intermediate image is formed by the objective lens system 1. Lens system 2 allows distance 2□ from the image-side focal point Fl of the eyepiece system.
It is designed to be formed at the position of

The microscope optical system I in Fig. 1 is used when analyzing a luminescent sample or an absorbing sample, but it is also used for excitation of a sample S, as in the high-sensitivity microscopic multi-wavelength spectrometer of the present invention shown in Fig. 2. A sample S equipped with a light source Ls and irradiated by the excitation light source Ls
An enlarged image of fluorescence, Raman radiation, etc. may be formed. This makes it possible to perform fluorescence analysis, Raman analysis, etc. of a sample in a local area. In this case, as the excitation light source Ls, a known excitation light source used for fluorescence analysis, Raman analysis, etc. can be used, but in particular, a fixed wavelength laser or a wavelength selectable one can be used.

The spectroscopic detection optical system (2) is an optical system that confines light from a minute area or a certain extent of spread area of the enlarged image sb of the sample S formed by the microscope optical system I to a point or line and emits it. System 3: This point-like or linearly limited position is set as a focal point, and the light emitted from it is taken in as much light as possible and made parallel by collimator lens 4 with a small F number. A spectrometer consisting of a reflection type diffraction grating 5 that diffracts and separates light, and an imaging lens 6 that images the parallel light separated by the reflection type diffraction grating 5 as a spectral image on an image plane P, and a spectral image. It is composed of a highly sensitive one-dimensional or two-dimensional photodetector 7 arranged on a surface. A computer 8 is connected to the output side of the highly sensitive one-dimensional or two-dimensional photodetector 7, and performs spectrum analysis based on the output from the computer 8. Incidentally, the reflection type diffraction grating 5 is configured to be rotatable as shown by the arrow shown in the figure, centering on a point C, in order to adjust the detection spectrum range.

In this case, the optical system 3 for emitting light from a minute region of the enlarged image sb of the sample or a region having a certain degree of spread, in a dotted or linear form, is used to There is no particular restriction as long as the light can be extracted in the form of points or lines. For example, the third
As shown in the spectral detection optical system (3), a pinhole or slit 3a is arranged on the image plane of the enlarged image sb, and the enlarged image S
The light from b may be narrowed down, or the fourth
Like the spectral detection optical system H shown in the figure, it may be configured with a light concentrator 3b that collects the light from the enlarged image sb from a condensing window and emits it to a point-like or linear exit end. .

However, the optical system 3 that confines the enlarged image Sb to a minute region or a region with a certain degree of spread in the form of a point or a line.
The size of the hole depends on the size of the hole. For example, when the hole is a round hole with a diameter, the light beam enters the spectroscope as a light beam with a wide diffraction angle of λ/D. That is, in order to increase the spatial resolution of the enlarged image sb, if the diameter of the hole is made smaller, the diffraction angle becomes larger, and the beam becomes a spread beam and enters the spectroscope. on the other hand,
When the diameter of the hole is increased, the diffraction angle becomes smaller and the beam spreads out as determined by the lens system of the microscope, so the loss of the beam is small, but the spatial resolution becomes worse. The selection of the diameter of the optical system 3 should be considered in order to improve sensitivity, depending on the spatial resolution and luminescence of the sample.

In such a high-sensitivity multi-wavelength spectrometer H, the grating interval of the diffraction grating 5 is d, the incident angle to the diffraction grating 4 is 1, the diffraction angle from the diffraction grating 5 is β, the wavelength is λ, and the diffraction When the order is m, the extremely weak incident light from the enlarged image sb of the sample S is diffracted so as to satisfy the relationship sin i + sin β = mλ/d, and is transmitted onto the highly sensitive one-dimensional or two-dimensional photodetector 7. A spectral image is formed on the
By analyzing the output from the highly sensitive one-dimensional or two-dimensional photodetector 7 using the computer 8 and determining the coordinates of the image and the image intensity at that point, bioluminescence, chemiluminescence, extremely weak fluorescence due to excitation light, etc. can be detected. It is possible to simultaneously measure the spectral characteristics of extremely weak light emitted from small objects. by the way,
In the above formula, so as to satisfy the relationship m--1,
The basic type (-1st order oblique incidence spectroscopy type) in which an imaging lens 6 and a high-sensitivity one-dimensional or two-dimensional photodetector 7 are arranged, and m-+
A basic type (+
There is a 1st-order oblique incidence spectroscopy type). FIG. 5(a) shows a -1st order oblique incidence spectroscopy type, and FIG. 5('b) shows a +1st order oblique incidence spectroscopy type. In these cases, a blazed diffraction grating is used as the reflection type diffraction grating 4, and is arranged as shown in the figure.

Let us consider the relationship between the apertures of the collimator lens 4 and the imaging lens 6 in the -1st order oblique incidence spectrometer shown in FIG. 5(a) and the +1st order oblique incidence spectroscope shown in FIG. 5(b). Figure 6 (
a) is an optical path diagram for studying the -1st order oblique incidence spectroscopy type, where the diameter of the collimator lens 4 is D1, the width of the diffraction grating is l, the diffraction angle of the center wavelength is βc1, and the diameter of the imaging lens 6 is D1. Di with diameter Do = j7 cos i D, = j'sin (2/π-βc,) = fflco
s βC.

By the way, in the case of this type, in order for the light to effectively reach the photodetector 7, since I<βc8, it is necessary to select the aperture of the imaging lens 6 so as to satisfy the condition of Di>Do. be. In the same way, if we consider the +1st-order oblique incidence spectroscopy shown in Figure 6, D, = ji! cos
1 Do=j! cosβC.

In this type of case, in order for light to effectively reach the photodetector 7, i>β. Therefore, it is necessary to select the aperture of the imaging lens 6 so as to satisfy the condition Di<Do. Based on this study, we will consider which type should be selected depending on the type of light to be separated. −
The first-order oblique incidence spectroscopy type uses a blazed diffraction grating as shown in Figure 6 (a).
), since the arrangement must be such that the incident light also hits a short diffraction surface, light reflected from this surface becomes stray light and increases background light. On the other hand, in the +1st-order oblique incidence spectroscopy type, as shown in Figure 6(b), the diffraction grating is arranged so that the light does not hit the short diffraction surface of the blazed diffraction grating, so stray light does not occur. However, as mentioned above, the aperture of the imaging lens 6 needs to be larger than that of the collimator lens 4. However,
In the highly sensitive multi-wavelength spectrometer used in the present invention, the aperture of the collimator lens 4 is as large as possible, so it is difficult to use a lens with a larger aperture as an imaging lens. Therefore, the +1st-order oblique incidence spectroscopy type can be said to have a large loss when the imaging lens 6 has a diameter similar to that of the collimator lens 4. As described above, it can be said that the -1st order oblique incidence spectroscopy type is suitable for the spectroscopic detection of extremely weak natural luminescence such as bioluminescence and chemiluminescence. On the other hand, in the spectroscopy of extremely weak light emitted by excitation light such as Raman scattering and fluorescence, where the influence of stray light is significant, the +1st-order oblique incidence spectroscopy type, in which stray light does not hide the signal light, although there is some loss, It can be said that this is more suitable. Of course, when designing the lens, you are free to use collimator lenses,
If the aperture and F number of the imaging lens can be achieved, then the +1st order oblique incidence spectroscopy type is better for the spectroscopic detection of extremely weak natural luminescence such as bioluminescence and chemiluminescence. I can do it. Also, when it is necessary to improve the resolution, a +1st order oblique incidence spectroscopy type is preferable.

In addition, as the collinator lens 4 and the imaging lens 6,
Not only a spherical lens with a small F number, but also an aspherical lens or a Fresnel lens may be used.

When performing spectroscopic analysis of the light from the enlarged image Sb of the sample using such a high-sensitivity multi-wavelength spectrometer H, the above-mentioned first to third
As shown in the figure, the diffracted light emitted from the imaging lens 6 includes 11th-order light, +1st-order light, etc. in addition to the 0th-order light, so as mentioned earlier, the diffracted light of an appropriate order is The imaging lens 6 is arranged so that the center wavelength is at the center of the highly sensitive one-dimensional or two-dimensional photodetector 7, and the angle of the diffraction grating 5 is adjusted.

By the way, as a modification of the basic type of high-sensitivity multi-wavelength spectroscopic device H shown in FIG. 1, a spectroscopic device can be considered in which a single lens serves as both a collimator lens and an imaging lens. As shown in FIG. 7(a), a collimator/imaging lens 9 with a small F number is placed in parallel in front of the reflective diffraction grating 5, and the entrance slit 3 is positioned at the focal point of this lens 9. A highly sensitive one-dimensional or two-dimensional photodetector 7 is placed at the image position of the diffracted light by the diffraction grating 5 by the dual-purpose lens 9, and the light from the sample is vertically incident thereon (vertical incidence spectroscopy type). The positions of the entrance slit 3 and the high-sensitivity one-dimensional or two-dimensional photodetector 7 may be exchanged so that the light from the sample is incident from an oblique direction as shown in FIGS. Grazing incidence spectroscopy type). The oblique incidence spectroscopy type shown in figure (b) has better angular resolution of diffracted light than the normal incidence spectroscopy type shown in figure (a). The spectroscopic type is excellent, and the highly sensitive one-dimensional or two-dimensional photodetector 7 is a combination of a two-dimensional photon counter and a low-afterimage vidicon (VIMS), These include a photon counting type image measurement system (PIAS) as shown in FIG. 9, an array photodetector in which diode photodetectors are arranged in an array, a CCD, and the like.

To briefly explain VIMS and PIAS among these, in FIG. The light enters a microchannel plate (MCP) 25 connected in two stages, is amplified, and hits a fluorescent screen 26 on the output surface to form a bright spot. This bright spot is imaged by the lens 27 on the photocathode of the low afterimage vidicon 28, and the two-dimensional position of the bright spot to which the photon corresponds is determined from the output of the vidicon 28 as a pulse signal, so the distribution of this bright spot is obtained. By doing this, an ultra-weak light spectral distribution image can be obtained.

In addition, in the PIAS shown in FIG.
The configuration up to the CP25 is the same as that in Fig. 8 (although the MCP25 in Fig. 9 has a three-stage connection).
The electron group emitted from the MCP 25 enters the silicon semiconductor device detector (PSD) 29 placed after it, is further amplified by the electron impact effect, and is output as a pulse signal to the PSD.
It is output from 29. The PSD 29 is a charge distribution type position detector that has four signal output electrodes 30 around it.
Charges generated inside the PSD 29 are distributed to these four electrodes 30 according to their generation positions via the resistance layer on the surface. As a result, a signal corresponding to the position of the center of gravity of the electron group incident on the PSD 29, that is, the position of the bright spot, is obtained from the four electrodes 30. The pulse signal obtained from the PSD 29 is amplified by an amplifier 32 and then guided to a position calculation device 31.

Here, these pulse signals are integrated by an integrating circuit 33 to determine the amount of charge from each electrode 30. Next, these signals are led to an addition/subtraction circuit 34, and then to a divider 36 via a wind gate 35 to be converted into a position signal, which is then AD-converted by an AD converter 37 and output.

This output signal is processed to determine the distribution of bright spots, and an ultra-weak light spectrum distribution image can be obtained. Furthermore, Figure 8,
In FIG. 9, the symbol. indicates an objective lens that images incident photons (arrows) onto the photocathode 22.

By the way, high-sensitivity multi-wavelength spectrometers (2) are not limited to the ones shown in Figures 1 to 7, but can also be used by arranging two or more spectrometers in multiple stages and converting the angular dispersion into an additive dispersion array by using the diffracted light several times. By lowering the resolution, it is also possible to use one with improved overall resolution. Of course, you can also array the difference variance. An example of the configuration of such a multiplexed multi-wavelength spectrometer is shown in FIG. 1O. In the figure (
a) is the −1st order oblique incidence spectrometer A1 in FIG. 5(a).
~A, are arranged in four stages in series to form a rectangular structure as a whole, and the same < (b) is shown in Fig. 5 (b).
The +1st order oblique incidence spectrometers A1 to A4 are arranged in series in four stages to form a rectangular structure as a whole, (C)
The device is constructed by arranging +1st order oblique incidence type spectrometers A1 to A in three stages in series. Note that the number of stages is not limited to the above.

As described above, in the high-sensitivity microscopic multi-wavelength spectrometer of the present invention, the optical system 3 is configured to limit and emit a minute region or a region with a certain degree of spread of the enlarged image sb in the form of a point or a line;
The highly sensitive multi-wavelength spectrometer ■, which has a collimator lens 4 with a small F number, a reflective diffraction grating 5, an imaging lens 6, and a highly sensitive one-dimensional or two-dimensional photodetector 7, uses bioluminescence, chemiluminescence, and excitation light. The following excellent effects are achieved in spectroscopic analysis of extremely weak light from an enlarged image sb of a minute object exhibiting extremely weak fluorescence or the like. That is, since the optical system 3 makes the light from the enlarged image sb enter the collimator lens 4 as a point or linear light source, the collimator lens 4 causes the light from the enlarged image sb to enter the collimator lens 4.
The light from b is made into parallel light beams with high parallelism without leaking and is made incident on the reflection type diffraction grating 5. Therefore, the reflection type diffraction grating 5 fully exhibits its resolution and can separate extremely weak light. Furthermore, since the collimator lens 4 and the imaging lens 6 can be arranged very close to the reflective diffraction grating 5, the device can be made smaller. Furthermore, since the collimator lens 4 and the imaging lens 6 can use bright lenses with the smallest possible F number, the brightness of the entire device, which is a composite thread of both lenses, can be sufficiently increased. By combining with a highly sensitive one-dimensional or two-dimensional photodetector 7, it becomes possible to simultaneously analyze multiple wavelengths of extremely weak light such as bioluminescence, chemiluminescence, and extremely weak fluorescence caused by excitation light, which has been difficult in the past.

〔Effect of the invention〕

As described above in detail, the high-sensitivity microscopic multi-wavelength spectrometer of the present invention has bioluminescence, bioluminescence,
It uses a high-sensitivity multi-wavelength spectrometer that can easily perform multi-wavelength simultaneous spectroscopic analysis of ultra-weak light from minute objects exhibiting chemiluminescence, ultra-weak fluorescence due to excitation light, etc.
It is capable of detecting extremely weak light, is compact and has excellent operability, and is particularly effective as a means for researching living organisms using bioluminescence, chemiluminescence, extremely weak fluorescence caused by excitation light, etc.

[Brief explanation of drawings]

FIG. 1 is an optical layout diagram of one embodiment of the high-sensitivity microscopic multi-wavelength spectrometer of the present invention, FIG. 2 is an optical layout diagram of another embodiment, and FIG.
4 is an optical layout diagram of an embodiment of a high-sensitivity multi-wavelength spectrometer used in the high-sensitivity multi-wavelength spectrometer of the present invention; FIG. 4 is an optical layout diagram of another embodiment of the high-sensitivity multi-wavelength spectrometer; Figure 5 is an optical layout diagram of the two basic types of the high-sensitivity multi-wavelength spectrometer shown in Figure 3, Figure 6 is an optical path diagram for examining the basic type shown in Figure 5, and Figure 7 is the high sensitivity of the present invention. An optical layout diagram of another embodiment of a highly sensitive multi-wavelength spectrometer used in a microscopic multi-wavelength spectrometer, FIG. 8 is a sectional view of a combination of a two-dimensional photon counter and a low-afterimage vidicon used in the present invention, and FIG. 9 10 is a cross-sectional view of another photon-counting image measuring device, FIG. 10 is an optical arrangement diagram of a highly sensitive multiplexed multi-wavelength spectrometer used in the present invention, and FIG. 11 is a block diagram of a conventional Raman microscope system. S: Sample, ■: Microscope optical system, ■: High-sensitivity multi-wavelength spectrometer, 1: Objective lens system, 2: Eyepiece system, Sa: Intermediate image of sample, Sb: Enlarged image, Ls: Excitation light source, spectroscopy Instrument A I""A 4: Spectrometer, 3: Optical system that confines light from a micro region of an enlarged image of the sample or a region with a certain degree of spread to a point or line and emits it, 3a: Input slit, 3b: light concentrator, 4: collimator lens, 5: reflection type diffraction grating, 6; imaging lens, 7;
Highly sensitive one-dimensional or two-dimensional photodetector, 8: computer,
9: Collimator/imaging lens, 21: Two-dimensional photon counter, 22: Photocathode, 23: Mesh, 24: Electron lens, 25: Microchannel plate (MCP)
, 26: Fluorescent screen, 27: Lens, 28: Low afterimage vidicon, 29: Silicon semiconductor device detector (PSD), 3
0: Signal output electrode, 31: Position calculation device, 32: Amplifier, 33: Integrating circuit, 34: Addition/subtraction circuit, 35: Wind gate, 36: Divider, 37: AD converter, Lo:
objective lens

Claims (7)

[Claims] (1) A microscope optical system that forms an enlarged image of a sample, and an optical system that confines light from a minute area or a certain amount of spread area of the enlarged image of the sample to a point or line and emits it; A bright collimator lens focuses the light emitted from the point-like or linear exit end of the optical system and makes it parallel.Reflection-type diffraction that diffracts and separates the light made parallel by the collimator lens. An imaging lens that forms parallel light separated by a grating or a reflective diffraction grating as a spectral image on an image plane, and a highly sensitive one-dimensional or two-dimensional photodetector placed on the image plane of the imaging lens. 1. A high-sensitivity microscopic multi-wavelength spectrometer comprising: a high-sensitivity multi-wavelength spectrometer comprising: (2) The microscope optical system has a wavelength-selectable sample excitation light source and is configured to form an enlarged image of re-emitted light from the sample irradiated by the excitation light source. The high-sensitivity microscopic multi-wavelength spectrometer according to claim 1. (3) A highly sensitive multi-wavelength spectrometer uses a blazed diffraction grating as a reflection type diffraction grating for spectroscopic detection of extremely weak natural luminescence such as bioluminescence and chemiluminescence, and each component of the spectrometer is 2. The high-sensitivity microscopic multi-wavelength spectrometer according to claim 1, characterized in that the device is arranged so that the next diffracted light can be taken out as a spectral image. (4) The high-sensitivity multi-wavelength spectrometer uses a blazed diffraction grating as a reflective diffraction grating for the spectroscopic detection of extremely weak light emitted by excitation light such as Raman scattering and fluorescence, and each component of the spectrometer has +1 order. 3. The high-sensitivity multi-wavelength microspectroscopy apparatus according to claim 1, wherein said multi-wavelength spectrometer is arranged such that the diffracted light can be taken out as a spectral image. (5) A high-sensitivity multi-wavelength spectrometer uses one dual-purpose lens as both a collimator lens and an imaging lens, and this dual-purpose lens is arranged parallel to the front surface of a reflection type diffraction grating,
On the focal plane of the dual-purpose lens 7, position the dot-shaped or linear exit end of the optical system that confines light from a minute area of the magnified image of the sample or an area with a certain degree of spread to a dot-shaped or linear shape and emits it. Claim 1 or 2, characterized in that a highly sensitive one-dimensional or two-dimensional photodetector is arranged on the image plane of the dual-purpose lens.
High-sensitivity microscopic multi-wavelength spectrometer described. (6) A high-sensitivity multi-wavelength spectrometer uses a slit or pinhole as an optical system to limit and emit light from a minute area or a certain extent of spread in an enlarged image of a sample in a dot or line shape. Claims 1 to 5 characterized in that
The high-sensitivity microscopic multi-wavelength spectrometer according to any one of the above. (7) The high-sensitivity multi-wavelength spectrometer uses a light concentrator as an optical system that confines light from a microscopic region of an enlarged image of the sample or a region with a certain degree of spread to a point or line. The high-sensitivity microscopic multi-wavelength spectrometer according to any one of claims 1 to 5. (8) The high-sensitivity multi-wavelength spectrometer limits the light from a minute region or a region with a certain degree of spread of an enlarged image of the sample to a point or line according to any one of claims 1 and 3 to 7. an optical system, a collimator lens, a reflection type diffraction grating, and
By arranging two or more spectrometers consisting of imaging lenses in multiple stages of the same type or different types, and arranging them for additive or differential dispersion, it is possible to control angular dispersion using diffracted light several times. Features a high-sensitivity microscopic multi-wavelength spectrometer.