Spectrofluorophotometer, spectrofluorometric measurement method, and image capturing method

Abstract

To provide a spectrophotofluorometer that obtains both spectra and sample images in the fluorescence measurement of a liquid sample.SOLUTION: A spectrophotofluorometer includes: a light source 11; an excitation-side spectroscope 12 that separates light of the light source 11 to generate excitation light; a fluorescence-side spectroscope 15 that separates fluorescence emitted from a sample S irradiated with the excitation light into monochromatic light; a sample container installation unit 19 for holding a sample container that is made of a transparent material and accommodates a liquid sample; a detector 16 that detects fluorescence emitted from the liquid sample; and an imaging device 20 that takes a sample image of the sample emitting the fluorescence. The sample container installation unit 19 includes: a port P1 for passing the excitation light; a port 3 for passing the fluorescence emitted from the sample; and a port P4 for the imaging device to observe the sample.SELECTED DRAWING: Figure 6A

Description

[Technical field]

[0001] The present invention relates to a spectrofluorophotometer, a spectrofluorometric measurement method, and an image capturing method. [Background technology]

[0002] A spectrofluorophotometer is a device that analyzes substances contained in various samples by irradiating the samples with excitation light and measuring the fluorescence emitted from the samples. That is, in a spectrofluorophotometer, the sample is irradiated with excitation light, the fluorescence emitted from the sample is dispersed, and spectral data such as the excitation spectrum, fluorescence spectrum, time change, and three-dimensional fluorescence spectrum are obtained. The excitation light is emitted over an area (generally 1 to 2 cm2) that depends on the optical system. 2 The spot is illuminated with a spot-like light (approximately 100 nm) and the fluorescence emitted from that spot is detected.

[0003] Patent Document 1 discloses a fluorescence spectrophotometer equipped with an integrating sphere and an imaging device, which simultaneously measures the spectrum of a sample and captures an image.

[0004] Patent Document 2 discloses a method for observing a sample from the side of the sample held on a flat substrate using an imaging device as a spectrophotometer for measuring the absorbance of a droplet sample. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2019-020362 A [Patent Document 2] JP 2006-258538 A Summary of the Invention [Problem to be solved by the invention]

[0006] Conventionally, in a typical spectrofluorometer, the fluorescence spectrum when irradiated with excitation light or the change in fluorescence intensity when the wavelength of the excitation light is changed is acquired as an excitation spectrum. In this case, the sample chamber in which the sample is placed must be a dark room, so it is difficult to confirm the state of the sample's fluorescence emission color, emission intensity, etc. when irradiated with excitation light.

[0007] The spectrofluorophotometer disclosed in Patent Document 1 captures an image of a sample during spectrum measurement and observes the distribution of the emission state in the plane, so that it is possible to obtain an image of the sample when irradiated with an arbitrary excitation wavelength. The disclosed device is equipped with an integrating sphere and an imaging device, and a sample is placed near the opening of the integrating sphere. In general, the inner surface of the integrating sphere is coated with a white material with high reflectance, such as barium sulfate. In the case of a liquid sample, placing the sample near the opening of the integrating sphere is not suitable for application to liquid samples because there is a risk of the sample contaminating the inner surface of the integrating sphere. In addition, by using an integrating sphere, the excitation light irradiated to the sample and the fluorescence emitted from the sample are irradiated to the sample or detected by a detector after repeatedly reflecting or scattering on the inner surface of the integrating sphere, so the light amount of the excitation light irradiated to the sample and the fluorescence detected by the detector becomes relatively small.

[0008] The spectrophotometer disclosed in Patent Document 2 holds a droplet-shaped sample on a sample holding plate and observes the sample from the side with an imaging device. If this configuration is applied to a spectrofluorophotometer, the emission direction of the fluorescence emitted from the sample may change depending on the placement of the droplet sample and the size of the droplet, making it possible that accurate measurements cannot be performed.

[0009] The present invention has been made to solve the above-mentioned problems, and has an object to provide a spectrofluorophotometer that does not require a complex optical system and can easily measure spectra and capture images simultaneously, even when measuring liquid samples. [Means for solving the problem]

[0010] The spectrofluorophotometer of the present invention includes a light source, an excitation side spectroscope that disperses light from the light source to generate excitation light, a fluorescence side spectroscope that disperses fluorescence emitted from a sample irradiated with the excitation light into monochromatic light, a sample container mounting section for holding a sample container made of a transparent material that contains a liquid sample, a detector that detects the fluorescence emitted from the liquid sample, and an imaging device that captures a sample image of the liquid sample emitting fluorescence.

[0011] The sample container installation section has ports in a direction in which excitation light is incident and in a direction in which fluorescence is extracted, and further includes a port in another direction for the imaging device to capture the sample image.

[0012] In the present invention, for example, the imaging device is provided at a position toward the bottom of the sample container mounting section, and the imaging device directly captures fluorescence emitted from the sample from the bottom of the sample container mounting section to capture the sample image.

[0013] The spectrofluorometric measurement method using a spectrofluorophotometer of the present invention is a spectrofluorometric measurement method using a spectrofluorophotometer comprising a light source, an excitation side spectroscope that disperses light from the light source to generate excitation light, a fluorescence side spectroscope that disperses fluorescence emitted from a sample irradiated with the excitation light into monochromatic light, a sample container mounting section for holding a sample container made of a transparent material that contains a liquid sample, a detector that detects the fluorescence emitted from the liquid sample, and an imaging device that captures a sample image of the liquid sample that emits fluorescence, wherein excitation light is incident from a port provided in the sample container mounting section in the excitation light incident direction, the detector detects the fluorescence from a port provided in the sample container mounting section in the fluorescence emission direction, and the imaging device captures the sample image via a port provided in yet another direction in the sample container mounting section.

[0014] The image capturing method using a spectrofluorophotometer of the present invention irradiates a sample with excitation light, and after the sample emits fluorescence and phosphorescence, blocks the excitation light, detects only the phosphorescence emitted from the sample after the excitation light is blocked, and captures an image of the sample in synchronization with the detection timing of the phosphorescence. Effect of the Invention

[0015] According to the present invention, it is possible to simultaneously obtain a sample image and a spectrum of a liquid sample with a simple configuration. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 is a block diagram showing an embodiment of a spectrofluorophotometer according to the present invention. [Diagram 2] FIG. 2 is a diagram showing an excitation spectrum indicating the intensity of fluorescence versus excitation wavelength in a spectrofluorometer. [Diagram 3] FIG. 3 is a diagram showing a fluorescence spectrum indicating the intensity of fluorescence wavelengths in measurements using a spectrofluorometer. [Figure 4] FIG. 4 is a diagram showing a time-varying spectrum of the intensity of fluorescence at a specific wavelength corresponding to excitation light at a specific wavelength in a spectrofluorometer. [Figure 5A] FIG. 5A is a diagram showing a three-dimensional spectrum, which shows a three-dimensional fluorescence spectrum of excitation wavelength, fluorescence wavelength, and fluorescence intensity. [Figure 5B] FIG. 5B is a diagram showing a three-dimensional spectrum, specifically, a three-dimensional time-varying spectrum of time, fluorescence wavelength, and fluorescence intensity. [Figure 6A] FIG. 6A is a schematic perspective view showing the configuration of the sample container setting section. [Figure 6B] FIG. 6B is a schematic top view showing the configuration of the sample container setting part. [Figure 6C] FIG. 6C is a schematic side view showing the configuration of the sample container setting part. [Figure 6D] FIG. 6D is a schematic diagram showing the configuration of the sample container setting part, showing a top view during irradiation with excitation light. [Figure 6E] FIG. 6E is a schematic perspective view showing another configuration of the sample container setting section. [Figure 6F] FIG. 6F shows an example in which a port for capturing an image of a sample is provided on the side of the sample container installation portion. [Figure 7A]FIG. 7A is a diagram showing the configuration of the sample container mounting portion and the camera module, and shows a configuration in which the camera module directly observes the sample container mounting portion. [Figure 7B] FIG. 7B is a diagram showing the configuration of the sample container mounting part and the camera module, and shows a configuration in which the camera module indirectly observes the sample container mounting part via a mirror. [Figure 8A] FIG. 8A is a diagram showing an example of an image and spectrum of a sample, showing an image when irradiated with white light. [Figure 8B] FIG. 8B is a diagram showing an example of an image and spectrum of a sample, showing an image when illuminated with monochromatic light of an arbitrary wavelength. [Figure 8C] FIG. 8C is a diagram showing an example of an image and spectrum of a sample, and shows the fluorescence spectrum when irradiated with the monochromatic light. [Figure 9A] FIG. 9A is a diagram showing the configuration of the sample container setting part when irradiated with excitation light and an example of an image captured at that time, showing a top view of the sample container setting part. [Figure 9B] FIG. 9B is a diagram showing the configuration of the sample container setting part when irradiated with excitation light and an example of an image captured at that time, showing an image captured from the bottom direction of the sample container setting part. [Figure 9C] FIG. 9C is a diagram showing the configuration of the sample container setting part when irradiated with excitation light and an example of an image captured at that time, showing a side view of the sample container setting part from the fluorescence capture direction. [Figure 9D] FIG. 9D is a diagram showing the configuration of the sample container setting part when irradiated with excitation light and an example of an image captured at that time, showing an image captured from the side direction of the sample container setting part. [Figure 10] FIG. 10 is a diagram showing a white board to be placed on the sample container placement section. [Figure 11] FIG. 11 shows timing charts during phosphorescence measurement, where (a) is a timing chart of excitation light, (b) is a timing chart of fluorescence and phosphorescence, (c) is a timing chart of detection by a detector, and (d) is a timing chart of exposure by a camera module. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Preferred embodiments of a spectrofluorometer according to the present invention will now be described with reference to FIGS.

[0018] 1 is a configuration block diagram showing one embodiment of a spectrofluorometer according to the present invention. The configuration of the spectrofluorometer of this embodiment will be described with reference to FIG.

[0019] The fluorescence spectrophotometer 1 of the present embodiment is an apparatus that irradiates a sample with excitation light and measures the fluorescence emitted from the sample, and includes a photometer unit 10, a data processing unit 30 that is disposed within the photometer unit 10 and controls the photometer unit 10 to analyze the sample, and an operation unit 40 that performs input and output.

[0020] The photometer unit 10 includes a light source 11 that emits continuous light, an excitation side spectrometer 12 that disperses the light from the light source 11 to generate excitation light, a beam splitter 13 that disperses the light from the excitation side spectrometer 12, a monitor detector 14 that measures the intensity of a portion of the light dispersed by the beam splitter 13, a fluorescence side spectrometer 15 that disperses the fluorescence emitted from the sample into monochromatic light, a detector (fluorescence detector) 16 that detects the fluorescence emitted from the liquid sample as an electrical signal of monochromatic fluorescence, an excitation side pulse motor 17 that drives the diffraction grating of the excitation side spectrometer 12, a fluorescence side pulse motor 18 that drives the diffraction grating of the fluorescence side spectrometer 15, and a sample container mounting unit 19 that contains and holds the sample (liquid sample) S to be measured.

[0021] The data processing unit 30 comprises a computer 31, a control unit 32 arranged within the computer, and an analog-digital converter 33 that digitally converts the fluorescence from the sample. The operation unit 40 also comprises an operation panel 41 that inputs input signals necessary for processing by the computer 31, a display unit 42 that displays various analysis results processed by the computer 31, and an interface 43 that connects the operation panel 41 and the display unit 42 to the computer 31.

[0022] According to the measurement conditions input by the operator via the operation panel 41, the computer 31 outputs a signal to the excitation side pulse motor 17, which drives the excitation side pulse motor 17 to set the excitation side spectroscope 12 to the target wavelength position. Similarly, according to the measurement conditions, the computer 31 outputs a signal to the fluorescence side pulse motor 18, which drives the fluorescence side pulse motor 18 to set the fluorescence side spectroscope 15 to the target wavelength position. The excitation side spectroscope 12 and the fluorescence side spectroscope 15 have optical elements such as a diffraction grating or a prism having a predetermined slit width, and the excitation side pulse motor 17 and the fluorescence side pulse motor 18 are used as power to rotate the optical elements via drive system components such as gears and cams, enabling spectrum scanning.

[0023] The liquid sample S is dispensed into, for example, a 10 mm square cell and placed on the sample container placement section 19. At this time, excitation light is irradiated, and the fluorescence generated to the side at 90 degrees to the excitation light is measured.

[0024] Furthermore, in this embodiment, a camera module (imaging device) 20 is provided near the sample container installation section 19 (for example, below the sample container installation section 19). Unlike the detector 16 which detects an electrical signal of fluorescence from the sample and acquires the intensity of the spectrum, the camera module 20 is a device which images and acquires a sample image (an image of the fluorescence emitted from the sample) due to the fluorescence from the sample S which emits fluorescence. The camera module 20 can be a general device capable of capturing a sample image.

[0025] In general, the electrical signal of the fluorescence from the sample S obtained by the detector 16 is displayed by the display unit 42 in the form of various spectra indicating the intensity of the fluorescence. Figures 2 to 5 show examples of two-dimensional spectra including two axes or three-dimensional spectra including three axes, which can be obtained not only by the fluorescence spectrophotometer 1 of this embodiment but also by a general fluorescence spectrophotometer. Figure 2 shows an excitation spectrum as an example of a spectrum, Figure 3 shows a fluorescence spectrum as an example of a spectrum, Figure 4 shows a time-varying spectrum as an example of a spectrum, and Figures 5A and 5B show three-dimensional spectra as examples of a spectrum.

[0026] The excitation spectrum shown in Fig. 2 is a spectrum obtained by measuring the fluorescence intensity when the excitation wavelength of the excitation light is changed for a sample. The excitation side spectroscope 12 changes the excitation wavelength from the measurement start wavelength to the measurement end wavelength, and irradiates the sample with excitation light of each wavelength. Fluorescence of a specific wavelength is detected by the detector 16 via the fluorescence side spectroscope 15, which is set to a fixed wavelength at that time, and is input as signal intensity to the computer 31 via the analog-to-digital converter 33. The computer 31 (control unit 32) analyzes and processes this signal intensity, and generates a spectrum that can be displayed by the display unit 42.

[0027] As a measurement result, the display unit 42 displays a two-dimensional excitation spectrum of the excitation wavelength and the fluorescence wavelength as shown in Fig. 2. The spectrum (graph) in Fig. 2 shows the fluorescence intensity (arbitrary unit) when the excitation wavelength is changed at a specific fluorescence wavelength (e.g., 550 nm).

[0028] The fluorescence spectrum shown in Fig. 3 is a spectrum obtained by irradiating a sample with excitation light of a fixed wavelength and measuring the fluorescence intensity for each wavelength when the fluorescence wavelength is changed. The sample is irradiated with excitation light from the excitation side spectroscope 12, which is set to a fixed wavelength. The fluorescence side spectroscope 15 changes the fluorescence of the measurement target at that time from the measurement start wavelength to the measurement end wavelength, and the detector 16 detects the change in fluorescence for each wavelength, which is then input as signal intensity to the computer 31 via the analog-to-digital converter 33. The computer 31 (control unit 32) analyzes and processes this signal intensity to generate a spectrum that can be displayed by the display unit 42.

[0029] As a measurement result, the display unit 42 displays a two-dimensional fluorescence spectrum of the fluorescence wavelength and the fluorescence intensity as shown in Fig. 3. The spectrum in Fig. 3 shows the fluorescence intensity when the excitation light has a specific wavelength (e.g., 450 nm) and the fluorescence wavelength is changed.

[0030] The time-varying spectrum shown in Fig. 4 is a spectrum obtained by irradiating a sample with excitation light of a fixed wavelength and measuring the fluorescence intensity of the fixed wavelength every unit time. The excitation light from the excitation side spectroscope 12, which is set to a fixed wavelength, is irradiated onto the sample, and the fluorescence generated at that time passes through the fluorescence side spectroscope 15, which is also set to the fixed wavelength at that time, and the change in the fluorescence intensity over time is detected by the detector 16. This is input as a signal intensity into the computer 31 via the analog-to-digital converter 33.

[0031] As a measurement result, the display unit 42 displays a two-dimensional time-varying spectrum of the measurement time and the fluorescence intensity as shown in Fig. 4. The spectrum in Fig. 4 shows the result of detecting the intensity of fluorescence of a specific wavelength (e.g., 550 nm) corresponding to an excitation light of a specific wavelength. As time passes, the fluorescent substance in the sample changes and decomposes, and the intensity often decreases.

[0032] 5A shows a three-dimensional spectrum displayed by the display unit 42, and in particular a three-dimensional fluorescence spectrum. The fluorescence spectrum is measured when the excitation wavelength is fixed for the sample, and when the fluorescence spectrum scanning is completed, the fluorescence wavelength is returned to the measurement start wavelength, the excitation wavelength is driven by a predetermined wavelength interval, and the fluorescence spectrum at the next excitation wavelength is measured. The obtained fluorescence spectrum is stored in three dimensions of excitation wavelength, fluorescence wavelength, and fluorescence intensity, and by repeating this process until the excitation wavelength reaches the final wavelength, a three-dimensional fluorescence spectrum can be obtained. This spectrum can be said to be a combination of the excitation spectrum in FIG. 2 and the fluorescence spectrum in FIG. 3.

[0033] The obtained three-dimensional fluorescence spectrum is depicted in a simulated three-dimensional format such as a contour map or a bird's-eye view by connecting the same fluorescence intensities with lines. The excitation wavelength and fluorescence wavelength that form a peak in the contour line are the excitation wavelength suitable for the sample and the characteristic fluorescence wavelength, and the operator can easily grasp the fluorescence characteristics of the excitation wavelength and fluorescence wavelength within the measurement range of the sample. Such a three-dimensional fluorescence spectrum is useful in that it can obtain a lot of information, such as the number of fluorescent substance components in the sample and the identification of the components.

[0034] FIG. 5B shows another three-dimensional spectrum displayed by the display unit 42, particularly a three-dimensional time-varying spectrum. The fluorescence spectrum is measured when the excitation wavelength is fixed for the sample, and when the fluorescence spectrum scanning is completed, the fluorescence wavelength is returned to the measurement start wavelength, and after a certain time, the fluorescence spectrum at the same excitation wavelength is measured. The obtained fluorescence spectrum is stored in three dimensions of measurement time, fluorescence wavelength, and fluorescence intensity, and repeated until the set measurement time is reached, thereby obtaining a three-dimensional time-varying spectrum. This spectrum can be said to be a combination of the fluorescence spectrum in FIG. 3 and the time-varying spectrum in FIG. 4. The obtained three-dimensional time-varying spectrum is depicted in a simulated three-dimensional format such as a contour map or a bird's-eye view by connecting the same fluorescence intensities with lines. Such a three-dimensional time-varying spectrum is useful for obtaining the time change of the fluorescence spectrum.

[0035] In this embodiment, the excitation light dispersed by the beam splitter 13 is irradiated onto the measurement target sample S. At this time, a part of the excitation light passes through the sample S and travels straight ahead. Since the camera module 20 observes the fluorescence emitted from the sample S, it is preferable to avoid installing the camera module 20 in the same direction as the excitation light irradiating the measurement sample S, and for example, the camera module 20 is placed at a position where the sample container is observed from the side or bottom.

[0036] The emitted fluorescence is taken in by the fluorescence side spectroscope 15 and dispersed into monochromatic light, which is detected by the detector 16 and taken as signal intensity by the computer 31 via the analog-to-digital converter 33, with various analysis results being displayed on the display unit 42. Meanwhile, the fluorescence emitted in the other direction is imaged by the camera module 20 to obtain a sample image which is then displayed on the display unit 42.

[0037] 6A to 6E are diagrams showing an example of the configuration of a sample container setting section 19 that holds a sample container 5 such as a rectangular cell that contains a liquid sample, with Fig. 6A being a perspective view of the sample container setting section 19, Fig. 6B being a top view of the sample container setting section 19, and Fig. 6C being a side view of the sample container setting section 19 from the fluorescence capture direction (the direction seen from the port P3 side). The sample container setting section 19 of this example holds a sample container 5 made of a transparent material such as a rectangular cell on five surfaces, namely the bottom and side surfaces, and is provided with four ports (holes) P1 to P4 that penetrate the outer and inner surfaces of the sample container setting section 19. On each surface of the sample container setting section 19, the portions other than the ports P1 to P4 are made of a light-shielding material.

[0038] Port P1 and port P2 are provided on surfaces facing each other. Port P3 is provided to the side of ports P1 and P2 at a predetermined angle (90 degrees in this embodiment). Port P4 is provided on the bottom surface of the sample container installation section 19. Port P1, located opposite the beam splitter 13, is capable of transmitting the excitation light generated by the excitation light side spectrometer 12 and split by the beam splitter 13, and controls the irradiation range of the excitation light on the sample according to its shape. Port P2 is provided opposite to port P1, and is capable of transmitting the excitation light that has passed through the sample in a linear direction. Port P3, located opposite the fluorescence side spectrometer 15, and port P4, located on the bottom surface of the sample installation section 19, are capable of transmitting the fluorescence emitted from the sample.

[0039] In this configuration, when excitation light is incident from the direction of port P1, the excitation light in the irradiation range according to the shape and position of port P1 is irradiated onto the sample in the sample container mounting section 19. FIG. 6D is a top view of the sample container mounting section 19 showing the horizontal length (width) of the light beam when the excitation light is irradiated onto the sample container mounting section 19 in this configuration. For example, when the excitation light L1 of the light beam corresponding to the length of width W1 is irradiated onto the sample container mounting section 19, if a port P1 having a width W2 narrower than the excitation light L1 is provided, the width of the excitation light L2 irradiated onto the sample in the sample container mounting section 19 becomes the width W2 of the port P1. At this time, a part of the excitation light travels straight through the sample and passes in the direction of port P2. Since the excitation light that travels straight through the sample may be reflected by the inner wall of the sample container mounting section 19 and affect the fluorescence measurement, it is preferable that the size (width) of port P2 is the same as port P1 or larger than port P1. Fluorescence emitted from the sample in the excitation light irradiation range specified by port P1 passes through port P3 and is guided to the fluorescence side spectroscope 15, where the spectrum is measured.

[0040] FIG. 6E is a side view of the sample container setting section 19 from the fluorescence capture direction, showing the vertical length (height) of the light beam when the sample container setting section 19 is irradiated with excitation light in this configuration. In this configuration, when the sample container setting section 19 is irradiated with excitation light L3 of a light beam corresponding to the vertical length H1, if a port P1 having a vertical length H2 shorter than the excitation light L3 is provided, the vertical length of the excitation light L4 irradiated to the sample in the sample container setting section 19 becomes the vertical length H2 of the port P1. At this time, a part of the excitation light travels straight through the sample and passes in the direction of the port P2. Since the excitation light that travels straight through the sample may be reflected by the inner wall of the sample container setting section 19 and affect the fluorescence measurement, it is preferable that the size (vertical length) of the port P2 is the same as or larger than the port P1. The fluorescence emitted from the sample passes through the port P3 and is guided to the fluorescence side spectroscope 15, where the spectrum is measured. At this time, the fluorescence emitted from the sample is emitted from the range where the excitation light is irradiated on the sample. In order to increase the amount of fluorescence guided to the fluorescence side spectroscope 15, it is desirable that the position and size of port P3 correspond to the excitation light irradiation range specified by the position of port P1. In other words, it is desirable that the position and size of port P3 match the excitation light L4.

[0041] A sample container 5 such as a rectangular cell used in a spectrofluorophotometer has a transparent bottom, so that the fluorescence emitted from the sample passes through port P4 and is guided to the camera module 20, which captures an image of the sample. When a sample container 5 with a flat and transparent bottom is placed on the sample container placement section 19, the camera module 20 observes the flat surface, and a clear image can be obtained.

[0042] That is, the sample container installation section 19 has at least a port P1 in the excitation light incident direction and a port P3 in the fluorescence emission direction, and further has a port P4 in another direction for the camera module 20 to capture a sample image. In the example of Figures 6A to 6E, the port P4 is provided at a position toward the bottom of the sample container installation section 19, and the camera module 20 is also provided toward the bottom of the sample container installation section 19, and captures the fluorescence emitted from the sample S directly to capture the sample image.

[0043] The shape and dimensions of the sample container installation section 19, and the number, shape, and direction of each port do not need to be limited as long as the direction of capturing the fluorescence or the direction of the camera module 20 is not affected by the excitation light traveling straight through the sample. For example, FIG. 6F shows an example in which a port P4 for capturing an image of the sample is provided on the side of the sample container installation section 19. In this case, the port P4 is provided at a position opposite to the port P3, and the sample container 5 such as a rectangular cell is observed from the side. That is, the port P4 is provided at a position in the side direction of the sample container installation section 19, and the camera module 20 is also provided in the side direction of the sample container installation section 19, and the fluorescence emitted from the sample S is directly captured to capture the sample image.

[0044] In this configuration, the camera module 20 is composed of a lens that focuses on the excitation light irradiation range, an aperture for adjusting the amount of light, a long-pass filter for cutting unnecessary light, an image sensor, etc. The camera module 20 is controlled by a computer 31 of a data processing unit 30.

[0045] 7A and 7B are side views of the configuration of the sample container setting section 19 and the camera module 20 for observing the sample container and sample in the sample container setting section 19, seen from the fluorescence capture direction of the sample container setting section 19, with Fig. 7A showing a configuration in which the camera module 20 directly observes the bottom surface of the sample container setting section 19 via port P4, and Fig. 7B showing a configuration in which the camera module 20 observes the bottom surface of the sample container setting section 19 via port P4 and a mirror 100. For image capture, the mirror 100 is desirably made of a material with a mirror surface and high reflectance, such as aluminum (reflectance of 80% or more as a guideline).

[0046] According to the observation method of observing a sample using the fluorescence spectrophotometer 1 of the present invention, it is possible to simultaneously acquire an image of the sample and a fluorescence spectrum while continuously changing the wavelength of the excitation light in the excitation side spectroscope 12. In other words, since the excitation light is not interrupted but continuously changed, and an image of the sample directly taken can also be acquired, it is possible to perform precise observation.

[0047] 8A to 8C show an example of an image captured by the camera module 20 through the port P4 from the bottom surface of the sample container installation section 19 and a measured fluorescence spectrum in the measurement of the sample S in the sample container 5. By setting the light irradiated from the excitation side spectroscope 12 to be so-called zero-order light (excitation wavelength EX=0 nm), it is possible to irradiate the sample S with undispersed white light. FIG. 8A shows an image captured when the sample S is irradiated with white light. Meanwhile, FIG. 8B shows an image captured when the excitation side spectroscope 12 is adjusted to irradiate monochromatic light of an arbitrary wavelength (450 nm in FIG. 8B and FIG. 8C, excitation wavelength EX=450 nm) and the monochromatic light is irradiated to the sample S. In the obtained image, the part corresponding to the bottom surface of the sample container installation section 19 (the bottom surface other than port P4) appears black because it is shielded from light. Port P4 provided at the bottom of the sample container installation section 19 is light-transmitting, so it is possible to observe the state of the sample S contained in the sample container when it is irradiated with white light or monochromatic light of any wavelength. The fluorescence spectrum shown in FIG. 8C can be obtained by measuring the fluorescence emitted from the sample S and detecting the intensity distribution of the fluorescence at each wavelength corresponding to the monochromatic light. In the fluorescence spectrum in FIG. 8C, the horizontal axis represents the fluorescence wavelength EM (nm). At this time, the image of the sample S (FIG. 8B) captured by the camera module 20 at the same time as observing the fluorescence is an image based on the fluorescence emitted from the sample S.

[0048] Since the image observed by the camera module 20 changes depending on the shape of the white light irradiated on the sample S and the excitation light designated by the port P1, the shape of the port P1 of the sample container installation section 19 is changed according to the purpose to capture the image. FIG. 9A shows a top view of the sample container installation section 19 when the excitation light is irradiated from the port P1 direction having a width W3, and FIG. 9B shows an image captured by the camera module 20 from the port P4 in the bottom direction of the sample container installation section 19 at that time (i.e., the embodiment of FIGS. 6A to 6E). The width of the excitation light irradiated on the sample S is determined by the width W3 of the port P1. Since the fluorescence emitted from the sample S is emitted from the range irradiated with the excitation light, the obtained image is an image in which the fluorescence is emitted from the sample S from the range of width W4, which is the same as the width W3 of the port P1.

[0049] Fig. 9C shows a side view of the sample container mounting section 19 from the fluorescence capture direction when excitation light is irradiated from the direction of port P1 having a vertical length H3, and Fig. 9D shows an image captured by the camera module 20 from port P4 on the side opposite the fluorescence capture direction of the sample container mounting section 19 at that time (i.e., the embodiment of Fig. 6F). Since the fluorescence emitted from the sample S is emitted from the range irradiated with the excitation light, the obtained image is an image of fluorescence emitted from the sample S from a range of vertical length H4, which is the same as the vertical length H3 of port P1.

[0050] In Figures 9B and 9D, the width along which the fluorescence is emitted is represented by a straight line, but since the fluorescence emitted from the sample S is emitted in all directions, the image actually obtained will be an image that has bleeding outside the width and vertical length.

[0051] 9A to 9D show that the width W3 (width W4) and the vertical length H3 (vertical length H4) are adjusted, which means that these sizes can be adjusted according to the amount of sample S (size of sample container 5). For example, when sample S is a valuable substance and its usage amount (size of sample container 5) is limited, appropriate measurement can be performed by using a sample container installation part 19 with a small width W3 and vertical length H3. Also, with regard to FIGS. 9C and 9D, for example, when sample S is a material that can be phase-separated by reaction, the phase is separated into, for example, two layers by promoting the reaction in sample container 5, and when only the material of the bottom layer corresponds to the vertical length H4, it is possible to measure only the material of the bottom layer. Various sample container installation parts 19 with various values ​​of width W3 and vertical length H3 can be prepared in advance.

[0052] Fig. 10 is a side view from the fluorescence capture direction, showing a configuration in which a white plate 200 is installed on the top of the sample container installation section 19 (i.e., the embodiment of Figs. 6A to 6E) when observed from the bottom side of the sample container installation section 19. The white plate 200 is installed on the top of the sample container installation section 19, and its flat surface faces the port P4 on the bottom side of the sample container installation section 19. The white plate 200 is made of a white material with high reflectance, such as aluminum oxide or a fluororesin-based white plate, or the white material is applied to the surface. The white material does not need to constitute the entire white plate 200, and may be applied to only one plane of the white plate 200.

[0053] A part of the fluorescence emitted from the sample S is reflected by the white plate 200 installed on the upper part of the sample container installation part 19, passes through the sample S and is captured by the camera module 20, so that the brightness value of the captured image can be improved compared to the case where the white plate 200 is not installed. When this configuration is applied, in order to prevent the fluorescence emitted from the sample S from being absorbed and attenuated by the sample S itself, it is preferable that the distance D1 from the range where the fluorescence is emitted to the liquid level is as short as possible. In addition, in order for most of the fluorescence emitted from the sample to reach the white plate, it is preferable that the distance D2 from the liquid level of the sample S to the white plate 200 is as short as possible.

[0054] Next, a method for capturing an image of a sample using the spectrofluorophotometer 1 will be described. A typical spectrofluorophotometer is capable of measuring phosphorescence. The measurement of phosphorescence is a method of measuring only phosphorescence after temporarily blocking the excitation light irradiated on the sample, taking advantage of the fact that phosphorescence has a longer lifespan than fluorescence. FIG. 11 is a diagram showing an example of a timing chart of measurement for capturing an image using the spectrofluorophotometer 1 of this embodiment. In this control, the photometer unit 10, and in particular its computer 31, controls the operation timing of the excitation side spectroscope 12, the detector 16, and the camera module 20.

[0055] First, the computer 31 controls the excitation side spectroscope 12, and the excitation light from the light source 11 is irradiated onto the sample S in the form of pulses at an arbitrary time interval (FIG. 11(a)). For a time T immediately after irradiation with the excitation light, the sample S emits both fluorescence and phosphorescence, but after the excitation light is blocked, only phosphorescence, which has a long life, is emitted for a time T' (FIG. 11(b)). The computer 31 controls the detector 16 in accordance with the timing of time T', and by corresponding the time period (sampling gate) for detecting light, it is possible to measure only the phosphorescence (FIG. 11(c)). At this time, the computer 31 synchronizes the exposure timing of the camera module 20 with the detector 16 (FIG. 11(d)), making it possible to simultaneously measure the spectrum of the phosphorescent sample S and capture an image of the sample.

[0056] According to the above-mentioned method, it is possible to measure the phosphorescence emitted from the sample S and to capture an image of the sample using the phosphorescence. Phosphorescent substances are usually recognized by the naked eye as a mixture of fluorescence and phosphorescence, but by measuring the phosphorescence as described above, it becomes possible to evaluate the fluorescence and phosphorescence separately. In conventional measurements using a spectrofluorometer, phosphorescence is recognized only in the form of a spectrum, but by obtaining an image of the phosphorescence, it is possible to obtain color information directly and obtain more detailed information about the sample.

[0057] An example of the use of a spectrofluorophotometer is a method in which a specific reagent is dropped onto a sample in a sample container 5, the sample is stirred, and the time change in the spectrum is measured. In the spectrofluorophotometer 1 of this embodiment, in addition to the time change in fluorescence, it is possible to observe the time change in an image of the luminescence state. Generally, a stir bar is often used to stir the sample, which makes it difficult to observe the sample. According to the spectrofluorophotometer 1 of this embodiment, it is possible to observe the sample S by observing and imaging it from a different direction through the port P4, or by using other methods such as irradiating ultrasonic waves from the surroundings to stir the sample S.

[0058] The present invention is not limited to the above-described embodiment, and can be appropriately modified, improved, etc. In addition, the material, shape, size, numerical value, form, number, arrangement location, etc. of each component in the above-described embodiment are arbitrary as long as they can achieve the present invention, and are not limited. [Explanation of symbols]

[0059] 1. Spectrofluorometer 5. Sample container 10 Photometer section 11 Light source 12 Excitation side spectrometer 13 Beam splitter 14 Monitor Detector 15 Fluorescence side spectrometer 16 Detectors 17 Excitation side pulse motor 18 Fluorescent side pulse motor 19 Sample container installation section 20 Camera module (imaging device) 30 Data Processing Section 31 Computer 32 Control section 33 Analog-to-Digital Converter 40 Control section 41 Operation Panel 42 Display section 43 Interface 100 Mirror 200 white board P1 Port P2 Port P3 Port P4 Port S sample

Claims

1. A light source; an excitation side spectrometer that generates excitation light by dispersing light from the light source; a sample container setting section that holds a sample container made of a transparent material for containing a liquid sample at its bottom and sides, and allows the excitation light generated by the excitation side spectroscope to travel in a straight line and be irradiated onto the liquid sample, and allows the excitation light to pass through the liquid sample in the straight line direction of the excitation light; a fluorescence side spectrometer that separates the fluorescence emitted when the excitation light is transmitted through the liquid sample into monochromatic light; a detector for detecting fluorescence emitted from the liquid sample; an imaging device that captures a sample image of the liquid sample that emits the fluorescent light; A spectrofluorometer comprising: the bottom surface and the side surface of the sample container installation portion are made of a light-shielding material and have a plurality of ports penetrating the inner and outer surfaces of the sample container installation portion, the plurality of ports include a port through which the excitation light passes in an incident direction of the excitation light, a port through which the fluorescence passes in an emission direction of the fluorescence, and a port provided in a direction different from an excitation light transmission direction of the port through which the excitation light passes and a fluorescence transmission direction of the port through which the fluorescence passes, and used by the imaging device to capture the sample image; Spectrofluorometer.

2. 2. The spectrofluorophotometer according to claim 1, the imaging device is provided at a position in a bottom direction of the sample container installation part, The imaging device is a spectrofluorometer that captures the fluorescence emitted from the liquid sample directly from the bottom surface direction of the sample container installation portion to capture the sample image.

3. 2. The spectrofluorophotometer according to claim 1, the imaging device is provided at a position in a lateral direction of the sample container installation part, The imaging device is a spectrofluorometer that captures the fluorescence emitted from the liquid sample directly from a side direction of the sample container installation portion to capture the sample image.

4. A light source; an excitation side spectrometer that generates excitation light by dispersing light from the light source; a sample container setting section that holds a sample container made of a transparent material for containing a liquid sample at its bottom and sides, and allows the excitation light generated by the excitation side spectroscope to travel in a straight line and be irradiated onto the liquid sample, and allows the excitation light to pass through the liquid sample in the straight line direction of the excitation light; a fluorescence side spectrometer that separates the fluorescence emitted when the excitation light is transmitted through the liquid sample into monochromatic light; a detector for detecting fluorescence emitted from the liquid sample; an imaging device for capturing a sample image of the liquid sample emitting fluorescence; A spectrofluorometric measurement method using a spectrofluorometer comprising: the bottom surface and the side surface of the sample container installation portion are made of a light-shielding material and have a plurality of ports penetrating the inner and outer surfaces of the sample container installation portion, The excitation light is made to enter through a port provided in the sample container setting part in a direction in which the excitation light is entered; the detector detects the fluorescence by passing the fluorescence through a port provided in the sample container setting section in a direction in which the fluorescence is emitted; the imaging device captures the sample image through a port provided in the sample container installation section in a direction different from an excitation light transmission direction of a port through which the excitation light passes and a fluorescence transmission direction of a port through which the fluorescence passes. Spectrofluorometer.

5. An image capturing method using the spectrofluorophotometer according to claim 1, comprising: the excitation side spectroscope irradiates the liquid sample with the excitation light, After the fluorescence and phosphorescence are emitted from the liquid sample, the excitation side spectroscope blocks the excitation light; the detector detects only the phosphorescence emitted from the liquid sample after the excitation light is cut off; the imaging device captures the sample image in synchronization with the detection timing of the phosphorescence; Image capture method.

Images