JPH01102342A - Fluoro-microscopic spectral method and apparatus - Google Patents

Abstract

PURPOSE:To enable the measurement of the quantity of a fluorescent material in a unit volume, by scanning a sample two-dimensionally by a converged laser light. CONSTITUTION:In an optical system, a laser light oscillated from a laser light source 1 is introduced into a modulation element 3. In a detecting system, the laser light is subjected to amplitude modulation by a photoacoustic deflector in the element 3 so as to measure the intensity of fluorescence from a sample 10. A scanner mirror 6 receives a signal from CPU 18 by a driving power source 8 and projects same onto a condenser lens 9 while executing a two-dimensional scanning, so as to irradiate the surface of the sample 10 two-dimensionally by a micro-spot light. Moreover, a sample stage 7 is provided with a rough two-dimensional drive system, which enables the search of the sample 10 in the visual field of a microscope by a driving power source 11 in linkage to the CPU 18. On the other side, the detecting system is provided with photomultiplier tubes 16a-16c, whereby the intensity of a fluorescent light separated into its components by BPF 15a and 15b is converted into an electric signal, amplified 17 and introduced into the CPU 18. Besides, a control-processing system subjects a pulse generated from the CPU 18 to A/D conversion, giving a signal to the power source 8 for two-dimensional scanning.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a laser microspectroscopy method and apparatus for optically and spectroscopically detecting extremely small and trace amounts of substances, and in particular,
The present invention relates to a multi-wavelength analysis type fluorescence microspectroscopy and apparatus suitable for identifying and detecting fluorescent substances unevenly distributed in minute parts of various samples such as cells at high speed and with high sensitivity.

(Prior Art) A substance dyed with a light-sensitive fluorescent dye absorbs light irradiated from the outside and generates fluorescent light with a longer wavelength. Based on these properties, conventionally, a laser beam is used as a light source to point-irradiate a sample stained with a phosphor dye, and the emitted fluorescence is detected by a photodetector, allowing it to be detected in minute parts within the sample. A scanning laser fluorescence microscope that observes the size, shape, position, distribution, etc. of substances within a sample from the intensity distribution of unevenly distributed fluorescent light [Reference Ana1. Chem, Acta 163 231 (19
84), G., J., Brakenhaff et al.], the fluorescence emitted from the sample is detected by combining a spectrometer and a photomultiplier tube, or by combining a polychromator and an STr detector to detect the spectrum and intensity distribution of the sample. Various types of fluorescence microspectroscopy devices have been developed, such as quantitative fluorescence microscopes that quantitatively measure substances.

Below, these conventional fluorescence microscopy spectroscopy methods will be briefly explained.

FIG. 5 is a diagram showing the configuration of a scanning laser fluorescence microscope. The laser light from the laser light source 51 is passed through the objective lens 5.
Sample 5, which was squeezed into a minute spot in Step 2 and stained with a fluorescent dye
3. Point irradiation. The fluorescent light generated from the sample is collected by a condensing lens 5.
4, the light is focused and guided to a photodetector 55 such as a photomultiplier tube, and the detected photoelectric signal is displayed on a display 56 synchronized with the XY scanning of the sample. This device has a confocal type in which an objective lens 52 and a condensing lens 54 are placed equidistantly from each other, with the sample surface at the center, and when the sample is placed at the focal position of both lenses, the The intensity of fluorescent light can be clearly detected.

Moreover, FIG. 4 is a configuration diagram of a quantitative fluorescence microscope. Using an argon laser as a light source 41, it is introduced horizontally into the microscope optical system via a beam splitter 42, and the direction of the beam is converted to the vertical direction, and the beam is introduced into the optical system through the objective lens 41.
Spot irradiation is performed on the 44th surface of the sample stained with fluorescent light in step 3. Fluorescent light or reflected light from the sample is introduced into the microscope optical system, a reflecting mirror 45 provided in the beam path is rotated, and the light is focused by each lens 46 provided at the front stage, and then the spectroscope 47 or polychromator 48 to be introduced. When fluorescent light is introduced into the spectrometer, a grating provided in the spectrometer is rotated to separate the fluorescent light into spectra, and the intensity of each spectrum is detected by a photomultiplier tube 49 placed at the rear. On the other hand, if the fluorescent light is divided into the entire spectrum by a built-in grating introduced into the polychromator, a diode array consisting of a large number of light-sensitive elements or a TV camera head installed in the rear SIT detector 50 is used. The entire spectrum is detected simultaneously.

(Problems to be Solved by the Invention) In order to examine the pathological condition of the whole organism from living cells or to improve the efficiency of cell surgery, it is necessary to The amount etc. must be clarified. For example, calcium within living cells plays an important role in mediating external stimuli depending on its concentration, and it is possible to reliably detect weak fluorescence from trace substances that fluctuate rapidly within such living cells. Therefore, the above-mentioned conventional devices measure only the intensity of the fluorescent light emitted from the sample, the intensity of a single spectrum, or the entire spectrum, and only measure partial information from the substances present in the sample. This method measures rough information, and there is a problem in that it cannot immediately respond to temporal changes in substances within living cells. Therefore, conventional devices are not suitable for measuring precise amounts of substances distributed within cells, that is, measuring absolute concentrations. Also,
In many cases, the shape of materials under a microscope is not uniform;
Complex surface refraction deforms the laser spot light. There is a problem in that such changes in excitation light density cause errors when measuring the amount of fluorescent substance.

An object of the present invention is to measure the concentration of a fluorescent substance, that is, the state of the fluorescent substance within a unit volume, by capturing not only the intensity of fluorescent light due to laser excitation but also its spectrum information.

(Means for Solving the Problems) In order to solve the above problems, the present invention scans a sample two-dimensionally with a focused laser beam, and measures the fluorescent light intensities of different wavelengths obtained by this scanning. I did it like that.

Specifically, it includes a laser beam generating means, a light scanning means for two-dimensionally scanning a sample with the laser beam generated from the laser beam generating means, and a fluorescent light intensity of different wavelengths generated by scanning the sample with the laser beam. This can be realized by a fluorescence microscope spectrometer equipped with at least two light intensity detection means.

(Function) One of the different wavelengths is determined as a wavelength that depends on the existence of the substance to be observed, and the other is determined as a wavelength that does not depend on the existence of the substance to be observed, and calculation processing is performed based on the fluorescence intensity of these wavelengths. This allows absolute measurement of the concentration of the substance to be observed without being affected by fluctuations in the measurement state. The measurement data thus obtained can be displayed in relation to the two-dimensional scanning position of the excitation laser beam, and detailed distribution information of the substance to be measured can be obtained.

(Effects of the Invention) According to the present invention, the distribution of minute and trace substances can be measured with high accuracy and sensitivity regardless of the measurement conditions. Since measurements can be performed even with relatively weak excitation light, it is possible to measure substances whose state changes when irradiated with strong light.

Such substances include living cell white matter (calcium ions,
(hydrogen ions, proteins, etc.). It is also effective for measuring substances at low temperatures, especially when the state changes due to the thermal effect of light absorption. This includes optical measurements of superconducting materials.

(Example) Hereinafter, an example of the present invention will be described using the system configuration diagram shown in FIG.

This device consists of (1) an optical system that irradiates the sample with a laser microspot, (2) a detection system that converts the fluorescent light emitted from the sample into an electrical signal, and (3) a detection system that controls the optical system and generates the detection signal. It consists of three parts: a control/processing system consisting of a processing computer and electronics.

In the optical system (1), laser light 2 oscillated from a laser light source 1 is introduced into a modulation element 3. In the detection system (2), in order to measure the fluorescence intensity from the sample, a phase-sensitive detection method is used to detect the modulation component from the back of the detected signal. For this purpose, the laser beam is amplitude-modulated by a photoacoustic deflector in the modulation element 3. The modulation element is powered by its driving power source 4
Since it is linked to the optical system control computer 18, the signal from the computer is automatically modulated.

Modulation processing includes normal modulation using a fixed frequency and random modulation using irregular frequencies, and can be selected as appropriate. The amplitude-modulated laser beam is processed into a rectangular wave by a shutter 5, reflected by two scanner mirrors 6 on the x and y axes, and then introduced from below the sample stage 7 parallel to the optical axis of the fluorescence microscope. be done. The scanner mirror receives a signal from the optical system control computer with a drive power supply 8, projects it onto a condenser lens 9 while scanning in two dimensions, and uses a micro spot light to collect fluorescently stained live cells under a microscope. The surface of the sample IO is irradiated two-dimensionally. The sample stage 7 is equipped with a rough two-dimensional drive system, and the sample can be easily searched within the field of view of the microscope by a drive power source 11 that is linked to a computer. Since the fluorescent light emitted from the sample is weak, the fluorescent light is effectively focused by the objective lens 12. By arranging the condensing lens 9 and the objective lens 12 in a confocal manner with respect to the sample surface, efficient light condensation becomes possible. This concentrated fluorescent light is
Since the ultraviolet rays of the laser beam are also taken in at the same time, they are removed by an ultraviolet blocking filter 13 placed on the optical path. The fluorescent light travels on the optical axis, is split by first and second partial reflection mirrors 14a and 14b provided on the optical axis, passes through each bandpass filter 15, and then splits into first and second partial reflection mirrors 14a and 14b. Detector 1
6a.

16b and further transmitted through the two partial reflecting mirrors 14b, the fluorescent light enters a third detector 16C provided at the final end of the optical path. 2 bandpass filters 1
5a and 15t) each transmit only a specific wavelength of fluorescent light, so by arranging the three detectors described above, the configuration is the same as that of separating into a plurality of spectral components and detecting them.

In the detection system (2), three high-sensitivity, low-noise photomultiplier dusts 16a, , 16b, and 16c are arranged, and the intensity of the fluorescent light separated by the bandpass filters 15a and 15b is electrically detected. The signal is converted into a signal, amplified by an amplifier 17, and introduced into a computer 18.

The control/processing system (3) performs D/A conversion of pulses generated by the computer and provides signals to the scanner mirror drive power source for two-dimensional scanning. Furthermore, voltages corresponding to the rotation of the mirror are stored as coordinates.

Further, the signal of the modulation component obtained from the photomultiplier dust or the magnitude of the modulation frequency is stored in a computer in combination with the coordinates. The stored coordinates and the fluorescence intensity at each of these points are reconstructed by a computer and imaged on the TV monitor 19. Imaging methods include a two-dimensional display method based on density or pseudo to - based on the level of light intensity, and a three-dimensional display method using two axes of intensity and an x-y plane for the irradiation position.

These images are created for each spectral component, but it is also possible to calculate and synthesize images of multiple spectral components. For example, when a fluorescence spectrum changes shape depending on the concentration of ions unevenly distributed within a substance (for example, intracellular free calcium ions), four arithmetic operations are performed on the intensities of, for example, two components of each spectrum component, and the concentration distribution is imaged. can do.

The fluorescence microspectroscopy apparatus constructed in this manner is operated, for example, according to the flowchart shown in FIG.

First, (a) input the number of photomultiplier tubes (the number required to extract the spectral characteristics). (c) Specify the size of the area to collect data. (C) Decide whether to modulate the light intensity. If not modulated, capture fluorescence data while moving the scanner.

(For mountain modulation, decide the parameters of the photoacoustic deflector. (e) First, select the modulation method as "normal modulation" or "random modulation", (f), (set the parameters of the selected modulation method) (5) Drive the scanner mirror to determine the laser beam irradiation position.(i) While irradiating the modulated laser beam, 0) Input the fluorescence intensity into the computer and process it to determine the fluorescence at the irradiation point. Measure light intensity.

Next, return to (5) and move the irradiation point. Repeat these steps to scan the entire data area and complete the measurement.

In the case of non-modulation, laser beam scanning and data collection are performed in the same manner as in the case of modulation.

Figures 3A to 3C show measurement results according to the present invention.

This experiment was conducted to measure the concentration distribution of calcium contained in cultured carrot cells (protoplasts).

In general, the wavelength that characterizes the fluorescence emitted from a dye combined with calcium ions is λ1, and the wavelength that characterizes the fluorescence unique only to the dye is λ2. Here, let R be the fluorescence intensity ratio at wavelength λ and wavelength λ2. This fluorescence intensity ratio R
Using the following formula, the concentration of calcium ions in the solution can be determined.

In this equation, R1, is the value of the fluorescence intensity ratio R when there are no calcium ions in the solution, and R□8 is the value of the fluorescence intensity ratio R when calcium ions are saturated.

C is a coefficient determined experimentally.

Figure 3A shows the dye 1ndo- bound to calcium ions.
The wavelength λ, which characterizes the fluorescent light emitted from 1, is 42
This is a diagram showing the fluorescence intensity distribution at 1 nm, and Figure 3B shows the wavelength λ2 of 467 nm, which characterizes the fluorescence unique to pigments.
FIG. 3 is a diagram showing the fluorescence intensity distribution of m. Note that 325 nm was used as the wavelength of the laser light for fluorescence excitation. By substituting the fluorescence intensities of different wavelengths thus obtained into the above equation, the calcium concentration distribution shown in FIG. 3C was obtained. Comparing FIG. 3C with FIG. 3A, it can be seen that the present invention can measure trace substances with extremely high sensitivity.

[Brief explanation of the drawing]

Fig. 1 is a system configuration diagram for realizing the present invention, Fig. 2 is a flowchart of a computer used to operate the system shown in Fig. 1, and Figs. 3A and 3B are illustrations of carrot silk cultivation. Figure 3C is a diagram showing the measurement results of the fluorescence intensity distribution for different wavelengths carried out on the specimen, and was obtained by calculating the fluorescence intensity distribution shown in Figures 3A and 3B. Figure 4 is a diagram showing the calcium concentration distribution, Figure 4 is a block diagram of a conventional quantitative fluorescence microscope, and Figure 5 is a schematic diagram of a conventional scanning laser fluorescence microscope spectrometer. (Explanation of symbols) 1.51.41... Laser light source, 12.52
．． 43...Objective lens, 10.53.44...
...Sample, 55...Photodetector, 56...Display, 42...Beam splitter-145...
・Polychromator, 16a, 16b, 16c, 49-...Photomultiplier tube, 50...SIT detector, 2...Laser light, 3...Modulation Element, 4... Modulation element drive power supply, 5... Shutter, 6... Scanner mirror, 7... Sample stage, 8...・Scanner mirror drive power supply, 9...
... Condensing lens, 11 ... Sample stage driving power supply, 13 ... Ultraviolet light a [r filter, 14 ...
...partial reflecting mirror, 15a, 15b...bandpass filter,
17...Amplifier, 18...Computer 1 19...TV Monitor Figure 2 Procedural Amendment (Method) 63.2.15 Showa Year Month Date Commissioner of the Japan Patent Office Small Mr. Kunio Kawa Q
l.Indication of the case 1988 Patent Application No. 260819
No. 2, Title of the invention Fluorescence microspectroscopy and apparatus 3, Relationship with the person making the amendment Applicant name (679) RIKEN 4, Agent 5, Date of amendment order January 26, 1988 7 , Contents of correction Attachment of Tofuri

Claims (6)

[Claims] (1) Fluorescence microspectroscopy, in which a sample is illuminated with a focused laser beam in a two-dimensional scanning manner, and the fluorescence intensity of different wavelengths obtained by this scanning is measured. (2) The fluorescence microspectroscopy method according to claim (1), characterized in that the measured fluorescence intensities of different wavelengths are subjected to arithmetic processing. (3) The fluorescence microspectroscopy method according to claim (1), wherein the laser light is modulated. (4) A fluorescence microscope according to claim (2), characterized in that the signal intensity obtained by the arithmetic processing is displayed two-dimensionally in correspondence with the scanning position of the laser beam. Spectroscopy. (5) The fluorescence microspectroscopy method according to claim 2, wherein the display is performed using a color depending on the signal intensity. (6) Laser light generation means, light scanning means for two-dimensionally scanning the sample with the laser light generated from the laser light generation means, and detecting fluorescence intensities of different wavelengths generated by scanning the sample with the laser light. A fluorescence microspectroscopy device comprising at least two light intensity detection means.