JP6829466B2 - Spectral fluorometer - Google Patents

Description

The present invention relates to a spectrofluorometer that non-destructively measures a sample in a sample container.

A method for analyzing a sealing material, a liquid, or the like enclosed (contained) in a sample container is known (Patent Documents 1 to 3). Further, a spectrofluorometer using fluorescence is known from the viewpoint of high sensitivity and amount of information (Non-Patent Document 1).

Patent Document 1 discloses that a specially shaped container is used by a method of nondestructively measuring the gas composition in a sealed container. Patent Document 2 discloses that a liquid sample is placed in a test tube and the liquid sample is measured with an absorptiometer. Patent Document 3 discloses an apparatus for inspecting a liquid-filled product filled with liquids for defects in a container, contents, and accessories.

Japanese Unexamined Patent Publication No. 9-127001 Japanese Unexamined Patent Publication No. 2001-33387 Japanese Unexamined Patent Publication No. 2003-130805

Japan Spectroscopy Society Measurement Method Series 3 "Fluorescence Measurement-Application of Biological Science", pp. 45-77, January 20, 1983, Society Publishing Center, Inc.

The measurement and inspection methods shown in Patent Documents 1 to 3 are applied only to a predetermined container, and in particular, in Patent Document 2, it is necessary to open the sample each time the measurement is performed, which may reduce the analysis accuracy. .. In addition, there is a problem that it is not possible to handle sample containers having different shapes, and it is not possible to avoid measurement obstacles such as labels, barcodes, and markings attached to the sample containers.

An object of the present invention is to provide a spectrofluorometer capable of detecting the fluorescence of a sample in a sample container at an optimum position without any obstacle on the surface of the sample container in the light passage path (optical path).

The spectral fluorometer of the present invention holds a light source, an excitation side spectroscope that generates excitation light by dispersing from the light of the light source, and a sample container containing a sample to be measured, and irradiates the excitation light. A position adjusting mechanism capable of adjusting the position of the sample container and a detector for detecting the fluorescence emitted from the sample are provided so that the position and the sample container have a relatively predetermined positional relationship.

As one aspect of the spectral fluorometer of the present invention, for example, the position adjusting mechanism has an in-plane adjusting mechanism capable of adjusting the in-plane position of the sample container and a height adjustment capable of adjusting the height position of the sample container. It includes at least one of a mechanism and a circumferential adjustment mechanism capable of adjusting the circumferential position by rotating the sample container.

As one aspect of the spectral fluorometer of the present invention, for example, the position adjusting mechanism includes a rotary height adjusting mechanism capable of adjusting the height at the same time while rotating the sample container to adjust the position in the circumferential direction.

As one aspect of the spectrofluorescence meter of the present invention, for example, the position adjusting mechanism can adjust the position of the sample container so that the excitation light and the central portion of the cross section of the sample container coincide with each other.

As one aspect of the spectral fluorometer of the present invention, for example, it has a control unit that calculates the intensity of fluorescence detected by the detector, and the control unit controls the position adjusting mechanism based on the intensity of fluorescence. Then, the sample container is positioned.

The spectral fluorometer of the present invention detects scattered light or fluorescence at each installation position with a position adjustment mechanism for measurement obstructing factors such as markings and labels on different outer shapes of the sample container and the outer circumference of the sample container. By doing so, the sample container can be set at the optimum position for evaluating the fluorescence characteristics.

The block diagram which shows an example of the spectrofluorescence meter which concerns on this invention. An example of the first embodiment of the position adjusting mechanism of the spectrofluorometer according to the present invention is shown, (a) front view and (b) side view. An example of the second embodiment of the position adjustment mechanism of the spectrofluorometer according to the present invention is shown, (a) front view and (b) side view. The graph which shows the strength and time change by the difference in the height position of a sample container using the 1st Embodiment of a position adjustment mechanism. The graph which shows the strength and time change by the difference in the height position of a sample container using the 2nd Embodiment of a position adjustment mechanism. The graph which shows the intensity of fluorescence with respect to the excitation wavelength in the measurement of the spectrofluorometer according to this invention. The graph which shows the intensity of a specific fluorescence wavelength in the measurement of the spectrofluorescence meter which concerns on this invention. In the measurement of the spectrofluorometer according to the present invention, a graph showing the intensity of fluorescence of a specific length corresponding to excitation light of a specific wavelength.

Hereinafter, preferred embodiments of the spectrofluorometer according to the present invention will be described in detail with reference to FIGS. 1 to 8.

FIG. 1 is a block diagram showing an example of a spectral fluorometer according to the present invention. The configuration of the spectrofluorometer of the present embodiment will be described in detail with reference to FIG.

The spectral fluorescence photometer 1 of the present embodiment is an apparatus for measuring a sample housed in a sample container 100, and is arranged in a photometer unit 10 and a photometer unit 10 to measure a sample in the sample container 100. A position adjusting mechanism 20 for adjusting the sample container 100 to an appropriate position, a data processing unit 30 for controlling the photometer unit 10 to analyze a sample, and an operation unit 40 for inputting / outputting are provided.

The photometric unit 10 includes a light source 11, an excitation side spectroscope 12 that disperses from the light of the light source 11 to generate excitation light, a beam splitter 13 that disperses light from the excitation side spectroscope 12, and a beam splitter 13. A monitor detector 14 that measures the intensity of a part of the dispersed light, a fluorescence side spectroscope 15 that separates the fluorescence emitted from the sample into monochromatic light, and a detector that detects an electric signal of monochromatic fluorescence (fluorescence). The detector) 16 is provided with an excitation side pulse motor 17 for driving the diffraction grating of the excitation side spectroscope 12, and a fluorescence side pulse motor 18 for driving the diffraction grating of the fluorescence side spectroscope 15.

The position adjusting mechanism 20 includes a sample setting unit 21 for placing and holding the sample container 100 containing the sample to be measured, and a driving unit 22 for driving the sample setting unit 21.

The data processing unit 30 includes a computer 31, a control unit 32 arranged in the computer 31, and an A / D converter 33 that digitally converts fluorescence from a sample. Further, the operation unit 40 includes an operation panel 41 for inputting an input signal necessary for processing by the computer 31, a display unit 42 for displaying various analysis results processed by the computer 31, an operation panel 41, a display unit 42, and a computer. It includes an interface 43 that connects to 31.

2A and 2B show an example of the first embodiment of the position adjusting mechanism 20, where FIG. 2A is a front view and FIG. 2B is a side view. 3A and 3B show an example of the second embodiment of the position adjusting mechanism 20, where FIG. 3A is a front view and FIG. 3B is a side view. The position adjusting mechanism 20 will be described in detail with reference to FIGS. 2 and 3.

In the first embodiment shown in FIG. 2, a sample setting portion 21 for placing and holding the sample container 100 is provided on the upper portion of the position adjusting mechanism 20, and the sample setting portion 21 holds the bottom of the sample container 100. Has a substantially circular recess 21a and a holding portion 21b surrounding the recess 21a. In the present embodiment, the position adjusting mechanism 20 includes an in-plane adjusting mechanism 23, a height adjusting mechanism 24, and a circumferential direction adjusting mechanism 25, and the sample setting portion 21 can adjust the in-plane position of the sample container 100. It is fixed to the in-plane adjustment mechanism 23 (see the XY directions in FIG. 2A).

Further, the drive unit 22 of the position adjusting mechanism 20 is electrically connected to the control unit 32 of the computer 31, and in the first embodiment, it has a height pulse motor 22a and a circumferential pulse motor 22b. Then, the in-plane adjusting mechanism 23 is fixed on the height adjusting mechanism 24, and the height adjusting mechanism 24 moves in the height direction (see FIG. 2A and Z direction) by being driven by the height pulse motor 22a. This makes it possible to adjust the height position of the sample container 100. Further, the circumferential direction adjusting mechanism 25 is fixed on the height adjusting mechanism 24, and the circumferential direction adjusting mechanism 25 is rotated in the circumferential direction (see FIG. 2A θ direction) by being driven by the circumferential pulse motor 22b. , The circumferential position of the sample container 100 can be adjusted.

In the second embodiment shown in FIG. 3, the sample setting portion 21 and the in-plane adjusting mechanism 23 are the same as those in the first embodiment, but the position adjusting mechanism 20 includes the height adjusting mechanism 24 and the circumferential direction adjusting mechanism 25. It is provided with a rotary height adjusting mechanism 26 in combination with and. Further, the drive unit 22 is a pulse motor as in the first embodiment. The rotary height adjusting mechanism 26 is a mechanism that can rotate the sample container 100 to adjust the circumferential position and adjust the height at the same time. For example, the sample container 100 rotates spirally by expanding and contracting with a combination of screws. The drive mechanism is spirally threaded up and down, but there are also pneumatic and hydraulic mechanisms, and there is no particular limitation.

The above-mentioned position adjusting mechanism 20 is a mechanism for setting an optimum measurement site for the installation of the sample container 100. The sample container 100 is, for example, a vial bottle or an ampoule bottle, and a label or engraving indicating the contents of a sample such as a solution contained in the sample container 100 is provided on the surface of the sample container 100. In addition, there are various shapes, and the position adjusting mechanism 20 of the present embodiment is a powerful means for non-destructive inspection in order to accurately acquire the fluorescence identification of the sample in the unopened state of the sample container 100. That is, the position adjusting mechanism 20 minimizes the influence of measurement error on different shapes of the sample container 100, the loss of light amount due to labels and markings, and the influence of reduced reproducibility.

Using the position adjustment mechanism 20, the irradiation position of the excitation light generated by the excitation side spectroscope 12 and the sample container 100 have a relatively predetermined positional relationship (for example, the position where the detection of the detector 16 is maximized). An example of the procedure of the installation method and the adjustment method of the sample container 100 will be described.

(1) The sealed sample container 100 is placed on the sample setting portion 21 of the position adjusting mechanism 20. At that time, the holding portion 21b of the sample setting portion 21 is in peripheral contact with the outer periphery of the sample container 100, and the sample container 100 is placed on the sample setting portion 21 in a stable state. The sample container 100 is an ampoule bottle, a vial bottle, or the like in which a medicine or a sample is enclosed (contained), and is basically a transparent sample container 100 for transmitting excitation light and fluorescence, and is a colored sample container 100. Is not targeted.

(2) Next, the in-plane adjusting mechanism 23 adjusts the sample container 100 in the in-plane direction (XY directions) so that the center of the bottom surface of the sample container 100 and the center of the optical axis are aligned with each other.

(3) Then, after inserting the excitation light mask 50 between the beam splitter 13 and the position adjusting mechanism 20, the sample container 100 is irradiated with white light of the 0th order light (transmitted light). The 0th-order light is irradiated, and the position is detected by the scattered light. The 0th order light may be laser light or the like.

(4) The height adjusting mechanism 24 moves the sample setting portion 21 to the Z-axis origin in the height direction (Z-axis direction).

(5) At the Z-axis origin, the circumferential direction adjusting mechanism 25 rotates the sample setting unit 21 in the circumferential direction (θ direction), and measures the output of the detector 16 in the 0th order light.

(6) The height adjusting mechanism 24 changes the Z-axis, and the circumferential direction adjusting mechanism 25 rotates the sample setting portion 21 in the circumferential direction to measure the output of the detector 16. In the second embodiment, the adjustments (4) to (6) are performed by the rotary height adjusting mechanism 26 by simultaneously moving the height position and the circumferential direction (for example, spirally). For example, the number of Z-axis measurements is calculated from the height (input value) of the sample container 100 and the moving distance (input value) in the Z-axis direction, and the measurement is performed for the number of times.

The position where the maximum output value of the detector 16 is obtained is the optimum position for measuring the sample of the sample container 100 at the position in the height direction and the position in the circumferential direction, and the fluorescence generated from the 90-degree direction of the excitation light at that position. Can be measured in the optimum state. The most reliable position (optimal position) for the excitation light to pass through the sample is the center of the optical axis of the excitation light and the center of the cross section of the sample container 100 without any obstacles existing on the surface of the sample container 100 such as labels and markings. In particular, it is the position where the central part of the bottom surface coincides.

Although the 0th-order light has been described in the above-described embodiment, if the excitation wavelength and fluorescence wavelength of the sample to be measured in advance are known, the position may be detected at the optimum excitation wavelength and fluorescence wavelength of the sample. Further, when the excitation side spectroscope 12 that cannot use the 0th-order light is used, monochromatic light that absorbs less sample may be irradiated as the excitation light, and the detector 16 may detect the wavelength of the same monochromatic light. When the sample container 100 having the same shape can be prepared, the optimum position may be narrowed by rough adjustment using pure water or a fluorescent reagent. Furthermore, when pure water sealed in the sample container 100 is used, Raman scattering may be used for position detection.

The example in which the sample setting portion 21 of the present embodiment has a substantially circular recess 21a and a holding portion 21b surrounding the recess 21a is described, but it is sufficient if the sample container 100 can be stably placed. The shape is not limited to a circular shape, but may be a substantially elliptical shape or a substantially quadrangular shape, and any holding portion 21b capable of holding the outer periphery of the sample container 100 may be used. The present embodiment is not limited to the present embodiment, and the present invention includes holding, holding the drawing shape, holding the slide bar type, and the like.

Although the position adjustment mechanism 20 is driven in order to obtain the optimum position, the position adjustment may be an automatic type or a manual type. In the case of the automatic type, it is possible to control the position adjusting mechanism 20 based on the fluorescence intensity by using the control unit 32 that calculates the fluorescence intensity detected by the detector 16.

The height adjusting mechanism 24 has a screw feed type, a rack and pinion type, a jack type, and the like, and in the case of the automatic type, a stepping motor can also be used. The circumferential direction adjustment mechanism 25 has a mechanism such as a screw feed type, and in the case of the automatic type, a stepping motor can also be used. There are various mechanisms of the height adjusting mechanism 24, the circumferential direction adjusting mechanism 25, and the rotary height adjusting mechanism 26, and the mechanism is not limited to the mechanism method.

The optimum position is detected by the detector 16 installed in the 90-degree direction of the excitation light, but when measuring the transmittance, a transmission detector is installed in the 180-degree direction with the excitation light in the same manner. The optimum position may be detected by the flow. Further, a diffraction grating is mainly used for the excitation side spectroscope 12 and the fluorescence side spectroscope 15, and a photomaru is used for the detector 16, but a silicon photodiode or a photomaru is used for the transmission detector, and the fluorescence side spectroscope 15 is used. It does not have to be installed.

The excitation light mask 50 inserted between the beam splitter 13 and the position adjustment mechanism 20 is used for the purpose of reducing the excitation light flux in order to improve the position confirmation accuracy. The usual excitation light is about 10 mm in the horizontal direction and about 5 mm in the vertical direction so as to match the size of the 10 mm square cell. The excitation light flux can be reduced by inserting the excitation light mask 50 between the excitation light emitting lens and the sample container 100. The size is preferably 1/5 or less of the diameter of the sample container 100 (for example, when the outer diameter of the sample container 100 is 20 mm, it is about 4 mm). However, if the excitation luminous flux is too small, noise becomes large, so it may be better to set a lower limit (for example, about 2 mm).

Graphs of the results of examining the output of the detector 16 at each height in order to determine the optimum position for measuring the sample contained in the sample container 100 are shown in FIGS. 4 and 5. The vertical axis represents the intensity (I) and the horizontal axis represents the time (s), and six height positions from Z1 to Z6 were selected.

The sample container 100 was placed on the position adjusting mechanism 20 and rotated in the circumferential direction at each height position (Zn: n = 1 to 6) to graph the change in strength. FIG. 4 is a measurement by the position adjusting mechanism 20 in the first embodiment, and is a discontinuous graph placed at a change position of each height position Zn. Further, FIG. 5 is a position adjusting mechanism 20 in the second embodiment, and is a continuous graph even at a change position of each height position Zn. Based on the results of the graphs shown in FIGS. 4 and 5, the sample container 100 to be measured is at the height position of Z2, which has the highest intensity at the position θ in the circumferential direction of the peak (in the example, around 180 degrees). It is understood that the measurements are optimally made.

By the above method, the sample container 100 is continuously evaluated for stability in non-destructive inspection such as quality confirmation of the sample and discrimination of the pseudo sample at the optimum position for measurement by the position adjusting mechanism 20 of the present embodiment. Is possible. It is also possible to perform additional studies and other tests using the same sample.

Analysis is performed by the spectrofluorometer 1 of the present embodiment using the sample container 100 set at the optimum position by the position adjusting mechanism 20. An example of the analysis method will be described with reference to FIGS. 1 and 6 to 8.

The continuous light emitted from the light source 11 such as the xenon lamp of the spectrofluorometer 1 is separated as the excitation light by the excitation side spectroscope 12, passes through the beam splitter 13, and is placed on the sample of the sample container 100 installed in the position adjustment mechanism 20. Be irradiated. At this time, the light amount (light intensity) of the excitation light partially split by the beam splitter 13 is measured by the monitor detector 14, and the fluctuation of the light source 11 is corrected.

The fluorescence emitted from the sample is separated into monochromatic light by the fluorescence side spectroscope 15, and detected by the detector 16 as an electric signal according to the intensity of the fluorescence. The fluorescence intensity signal is converted into digital data via the A / D converter 33, captured as signal intensity by the computer 31, and the measurement result is displayed on the display unit 42.

The spectrofluorometer 1 of the present embodiment is a wavelength drive system, and the excitation side spectroscope 12 is moved to a target wavelength position by driving the excitation side pulse motor 17 via the control unit 32 by a command of the computer 31. It is set. Further, the fluorescence side spectroscope 15 is also set at a target wavelength position by driving the fluorescence side pulse motor 18 via the control unit 32 according to a command from the computer 31.

The excitation side spectroscope 12 and the fluorescence side spectroscope 15 use optical elements such as a diffraction grating and a prism, and are powered by the excitation side pulse motor 17 and the fluorescence side pulse motor 18, for example, by a mechanism such as a gear and a cam. , The spectrum can be scanned by rotating the diffraction grating or prism.

Generally, in the excitation spectrum for measuring the fluorescence intensity when the excitation wavelength of the excitation light is changed with respect to the measurement sample in the sample container 100, the excitation wavelength is changed from the measurement start wavelength to the measurement end wavelength by the excitation side spectroscope 12. , The measurement sample is irradiated with excitation light of each wavelength, the change in fluorescence of a specific wavelength is detected by the detector 16 via the fluorescence side spectroscope 15 set to the fixed wavelength at that time, and the A / D converter 33 is used. It is taken into the computer 31 as a signal strength via.

As a measurement result, the display unit 42 displays a two-dimensional spectrum of the excitation wavelength and the fluorescence intensity as shown in FIG. The graph of FIG. 6 shows the fluorescence intensity when the excitation wavelength is changed at a specific fluorescence wavelength.

Further, the fluorescence spectrum for measuring the fluorescence intensity for each wavelength when the measurement sample in the sample container 100 is irradiated with excitation light having a fixed wavelength and the fluorescence wavelength is changed is the excitation side spectroscopy set to the fixed wavelength. The measurement sample is irradiated with the excitation light from the device 12, the fluorescence at that time is changed by the fluorescence side spectroscope 15 from the measurement start wavelength to the measurement end wavelength, and the change in fluorescence for each wavelength is detected by the detector 16, A. It is taken into the computer 31 as a signal strength via the / D converter 33.

As a measurement result, the display unit 42 displays a two-dimensional spectrum of the excitation wavelength and the fluorescence intensity as shown in FIG. 7. The graph of FIG. 7 shows the result of detecting the fluorescence intensity in a wide wavelength band in which the excitation light has a specific wavelength.

On the other hand, the time-varying spectrum in which the measurement sample in the sample container 100 is irradiated with excitation light of a fixed wavelength and the fluorescence intensity of the fixed wavelength is measured every unit time is the excitation side spectroscope 12 set to a fixed wavelength. The measurement sample is irradiated with the excitation light from the above, the wavelength generated at that time is fixed at the wavelength of the fluorescence side spectroscope 15, the change in fluorescence intensity with time is detected by the detector 16, and the change in fluorescence intensity with time is detected via the A / D converter 33. Is taken into the computer 31 as a signal strength.

As a measurement result, the display unit 42 displays a two-dimensional spectrum of the excitation wavelength and the fluorescence intensity as shown in FIG. The graph of FIG. 8 shows the result of detecting the fluorescence intensity of a specific wavelength corresponding to the excitation light of a specific wavelength. The pattern is substantially constant if there is no change in the positional relationship of the sample container 100 or decomposition of the sample.

The present invention is not limited to the above-described embodiment, and can be appropriately modified, improved, and the like. In addition, the material, shape, size, numerical value, form, number, arrangement location, etc. of each component in the above-described embodiment are arbitrary and are not limited as long as the present invention can be achieved.

The spectrofluorometer according to the present invention can be applied to a field in which a sample container is measured at an optimum position when a sample in a sample container having a different shape and a label or a stamp is subjected to non-destructive inspection.

1 Spectral fluorescence photometer 10 Photometer part 11 Light source 12 Excitation side spectroscope 13 Beam splitter 14 Monitor detector 15 Fluorescence side spectroscope 16 Detector 20 Position adjustment mechanism 21 Sample installation part 22 Drive part 23 In-plane adjustment mechanism 24 Height Adjustment mechanism 25 Circumferential direction adjustment mechanism 26 Rotary height adjustment mechanism 30 Data processing unit 31 Computer 32 Control unit 40 Operation unit 100 Sample container

Claims (4)

Light source and
An excitation side spectroscope that generates excitation light by splitting from the light of the light source,
A position adjusting mechanism capable of holding the sample container containing the sample to be measured and adjusting the position of the sample container so that the irradiation position of the excitation light and the sample container have a relatively predetermined positional relationship. ,
A detector that detects the fluorescence emitted from the sample and
A control unit that calculates the intensity of fluorescence detected by the detector,
A spectrofluorometer equipped with
The position adjusting mechanism includes a height adjusting mechanism capable of adjusting the height position of the sample container and a circumferential adjusting mechanism capable of adjusting the circumferential position by rotating the sample container.
The control unit rotates the sample container by the circumferential direction adjusting mechanism at each height position adjusted by the height adjusting mechanism, and has the highest intensity at the circumferential position of the obtained fluorescence peak. A spectrofluorometer that detects the height position and positions the sample container . The spectrofluorometer according to claim 1.
A spectrofluorometer in which the position adjusting mechanism includes an in-plane adjusting mechanism capable of adjusting the in-plane position of the sample container. The spectrofluorometer according to claim 1.
A spectrofluorometer including a rotary height adjustment mechanism in which the position adjustment mechanism can rotate the sample container to adjust the circumferential position and at the same time adjust the height. The spectrofluorometer according to claim 1.
The position adjusting mechanism is a spectrofluorometer capable of adjusting the position of the sample container so that the center of the optical axis of the excitation light coincides with the center of the cross section parallel to the bottom surface of the sample container.

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