JPH10123055A - Fluorescence spectrophotometer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a means by which a position on a sample automatically giving a maximum fluorescence intensity is detected automatically as a suitable measuring position when the sample in which a fluorescent substance exists locally inside a sample face is measured by using a reflection-type sample holder. SOLUTION: A driving mechanism is installed in such a way that its position can be controlled along an axis 5 which is parallel with a reflecting face (a sample face) 1 and which is parallel with a face 4 formed by an excitation-side optical axis 2 and by a fluorescence-side optical axis 3, along an axis 7 which is parallel with the reflecting face 1 and which is directed to the normal-like direction of the face 4 formed by the excitation-side optical acid 2 and by the fluorescence-side optical axis 3 and along an axis 6 which is directed to the normal-line direction of the reflecting face 1. At this time, the axes are scanned, and positions and fluorescence intensities on the three axes 5 to 7 are monitored and stored. Thereby, an optimum measuring position, an image formation position, the undulations of the sample face 1 and the two-dimensional distribution of a florescent substance on the sample face 1 are obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is suitable for qualitative and quantitative analysis of solid samples by a spectrofluorometer which is carried out for the purpose of research in medicine, pharmacy, chemistry, biochemistry or quality control of products. Applied to measurement to obtain sensitivity.

[0002]

2. Description of the Related Art For the measurement of solid and powder samples, refer to Hitachi, Ltd. "Instruction Manual for Spectrofluorometer 65
0-0161 solid sample holder ". In this case, there is no means for moving the sample surface, and the only option is to change the installation position of the sample on the sample holder. Also, Hitachi, Ltd., “Instruction Manual F-3010 / F-4010 Type Thin-Layer Cutoma Attached Device for Spectrofluorometer”, Part.No.250-
No. 9942 describes a spectrofluorometer having a function of moving two axes parallel to the sample surface, but there is no description of a sample surface having a function of moving three axes in the normal direction.

[0003]

When a solid sample in which a fluorescent substance is spread on filter paper or a sample in which the fluorescent substance is localized on the sample surface is measured using a reflection type sample holder, the fluorescent substance is most often measured. The higher the density, the higher the luminous intensity. This position should be searched for qualitative analysis or the like. However, conventionally, since there is no sample moving mechanism, it is necessary to repeat installation and measurement of the sample on the sample holder by trial and error, which is complicated, and a suitable measurement position is not always found. The present invention provides a means for automatically detecting the position on the sample that gives the maximum fluorescence intensity automatically.

Further, when there is undulation on the sample surface, the excitation light beam of the spectrofluorometer forms an image at a specific position in the sample chamber. A case where no image is formed on the sample surface occurs, and it becomes impossible to obtain a suitable sensitivity. The present invention provides a means for automatically obtaining an appropriate sensitivity by irradiating the excitation light with an image always formed on the sample surface.

[0005] Further, the present invention provides a means for giving knowledge about the undulation and the distribution of the fluorescent substance on the sample surface.

[0006]

Means for Solving the Problems In a reflection-type sample holder for solid or powder, (1) parallel to a reflection surface (sample surface) and parallel to a surface formed by an excitation optical axis side optical axis and a fluorescence side optical axis. Axis, (2)
An axis parallel to the reflection surface (sample surface) and oriented in the normal direction of the surface formed by the excitation optical axis side and the fluorescence side optical axis, and (3) an axis oriented in the normal direction of the reflection surface. A drive mechanism capable of controlling the position along the axis is provided, and these are scanned, and the positions on the three axes and the fluorescence intensity (or the scattered light intensity) are monitored and stored, so that the optimal measurement position, imaging position, and sample are obtained. The two-dimensional distribution of the fluorescent substance on the surface of the sample and the undulation of the surface is obtained.

[0007]

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

FIG. 1 shows each axis for explaining the invented sample holder.

[0009] Excitation light is incident on the sample surface 1 from the direction of the optical axis 2. Fluorescence emitted from a position on the sample surface where the excitation-side optical axis 2 intersects with the sample surface 1 or scattered light on the sample surface is observed in the direction of the fluorescence-side optical axis 3. Here, the excitation side optical axis and the fluorescence side optical axis are on the same plane 4. Reference numerals 5 to 7 denote movement directions of the invented sample holder, 5 denotes an axial direction parallel to the sample surface (reflection surface) and parallel to a plane formed by the excitation light side and the fluorescence side optical axis, and 6 denotes a normal direction of the sample surface. 7 denotes an axial direction parallel to the sample surface and normal to the plane formed by the excitation light side and the fluorescence side optical axis.

The sample holder is provided with a mechanism capable of independent movement and position control in these 5 to 7 directions.

More specifically, a solid sample holder is placed on a stage in which each axial direction of an electric XYZ stage is arranged corresponding to 5 to 7 axial directions. As the electric XYZ stage, a stage that can be externally controlled from a PC or the like or that is programmable with respect to stage movement is used.

FIG. 2 shows an example in which the fluorescent substance is localized at a specific portion in the sample surface, such as when the fluorescent substance is spread on filter paper.

The fluorescent substance 8 is localized on the sample surface 1.
The axial direction is parallel to the five sample surfaces (reflection surface) and parallel to the plane formed by the excitation light side and the fluorescence side optical axis in FIG. 1, and the seven surface parallel to the sample surface and formed by the excitation light side and the fluorescence side optical axis. The sample surface is scanned by an axial drive mechanism oriented in the normal direction. Since the excitation-side optical axis and the fluorescence-side optical axis are fixed, the excitation light beam 9 on the sample surface relatively scans on the sample surface. Although there are various scanning paths, it is assumed here that scanning is performed along a path such as 10. During scanning, the position and the fluorescence intensity are constantly monitored, and the position giving the maximum fluorescence intensity is stored. After the end of scanning, the stage is automatically moved to a position giving the maximum fluorescence intensity. Through these processes, when measuring the surface reflected fluorescence, the optimal position can be automatically detected, while the optimal position has been set by trial and error in the past.

FIG. 3 illustrates an example in which the surface of an undulating solid sample is scanned with the sample holder in the direction normal to the sample surface (direction 6). Here, the excitation light is incident in the direction of 11. The position where the two straight lines 11 intersect is the condensing position of the excitation light, and the light emission position observed at 12 is at the same position. In a), due to the undulation of the sample surface,
Excitation of sample and detection of fluorescence are not performed at the optimal position. Here, the sample surface is scanned in the axial direction 6 and the position where the maximum fluorescence intensity is provided and the fluorescence intensity are stored, and after the sample surface is scanned, the sample surface is automatically set to the position where the maximum fluorescence intensity is provided. I do. At position b), the sample is excited most efficiently, allowing the most efficient fluorescence detection.

FIG. 4 shows an example of a sample in which the surface of the sample has undulations and the fluorescent substance is localized on the surface of the sample.
a) is a top view of the sample, and b) is a plan view. here,
For the sake of simplicity, it is assumed that the undulations on the surface of the sample have a planar shape with a certain gradient. The sample stage is scanned on the sample along the path 10 as shown in FIG. Of course, a route other than the route 10 may be followed. This scan is a scan along axes 5 and 7 in FIG.
At this time, (1) the stage is moved in a 5-axis direction or a 7-axis direction by a fixed distance, (2) the sample stage is scanned along the 6-axis in FIG. Giving the maximum fluorescence or scattered light intensity at the location 6
The position on the axis and its fluorescence intensity or scattered light intensity are stored. Then, the sample stage is moved in a fixed manner along the five or seven axes, and the processing from (2) to (3) is repeated. In this way, for the target range of the sample surface, the position on the sample surface (this is represented by coordinate values along the 5, 7 axes)
And the undulation of the sample surface corresponding to the position (this is represented by coordinate values along six axes) and the maximum fluorescence intensity or the scattered light intensity are stored. That is, the coordinate values along five axes in the row direction of the matrix, the coordinate values along seven axes in the column direction, the coordinate values on the six axes that give the maximum fluorescence intensity or the scattered light intensity to the elements of the matrix, or the maximum fluorescence intensity or Stores the scattered light intensity itself. In addition to the matrix format, the storage method may be an array format or storage of only the elements if the range in the row direction and the column direction and the stage movement step are known. This storage location may be on the memory of the control unit that controls the spectrofluorometer, or may be a hard disk,
It may be on a floppy disk or other storage medium.

FIG. 5 shows an example in which the stored data is three-dimensionally displayed. a) represents the coordinates along the 5 and 7 axes and the coordinate values on the 6 axes in FIG. 4, and gives knowledge on the undulation of the surface of the sample. b) is a graph showing the coordinates along the 5 and 7 axes and the fluorescence intensity, and gives information on the localization of the sample in the plane.

[0017]

According to the present invention, the detection of the optimum measurement position on the measurement surface of a solid sample and the detection of the optimum imaging state, which have conventionally relied on trial and error, are automatically performed. When the fluorescent substance is localized on the surface of the sample and the distribution of the fluorescent substance is localized on the surface of the sample, it is possible to obtain the knowledge about the shape in the case of the undulation.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram showing coordinate values on a sample holder.

FIG. 2 is an explanatory diagram illustrating a method of scanning a stage on a sample surface.

FIG. 3 is an explanatory view showing a measurement example of a sample having undulations on the surface.

FIG. 4 is an explanatory diagram illustrating a sample having undulations on its surface and localizing a fluorescent substance.

FIG. 5 is an explanatory diagram of a three-dimensional graph display of measurement results.

[Explanation of symbols]

1 ... sample surface, 2 ... excitation side optical axis, 3 ... fluorescence side optical axis, 4 ...
Surface, 5, 6, 7 ... axis.

Claims (5)

[Claims] A light source, an excitation-side spectroscope for dispersing light from the light source and irradiating the sample with monochromatic light, a fluorescence-side spectroscope emitted from the sample, a detector for emitting light of the fluorescence-side spectrometer, Reflection for measuring solid and powder samples in the sample chamber in a spectrofluorometer composed of a control unit that performs wavelength shift of the above two spectrometers, data acquisition from the detector, data display, processing, etc. In the holder of the sample cell of the shape, the axial direction parallel to the sample surface and parallel to the plane formed by the excitation light side and the fluorescence side optical axis, and the normal direction parallel to the sample surface and formed by the excitation light side and the fluorescence side optical axis A drive mechanism capable of position control is provided along the axial direction facing and the axial direction facing the normal direction of the sample surface, and irradiates an arbitrary position on the sample surface with excitation light, and a portion irradiated with the excitation light. Fluorescence spectrophotometer characterized by being able to detect fluorescence from a fluorescent light. 2. A spectrofluorimeter provided with a drive mechanism capable of controlling the position, wherein an axial direction parallel to a surface of a solid sample to be measured and parallel to a plane formed by an excitation light side and a fluorescence side optical axis, The sample surface is scanned in both axial directions parallel to and perpendicular to the surface formed by the excitation light side and the fluorescence side optical axis, and the position and fluorescence intensity on the sample surface are detected and stored. A spectrofluorometer characterized by detecting a position on a sample surface giving the maximum fluorescence intensity and automatically setting the sample position at a position giving the detected maximum fluorescence intensity. 3. A spectrofluorimeter provided with a drive mechanism capable of controlling the position, wherein, when an arbitrary position on the surface of the solid sample is irradiated with excitation light, the sample surface is scanned in a direction normal to the sample surface. Detecting and storing the amount of movement and the fluorescence intensity, detecting the position giving the maximum fluorescence intensity after scanning, and automatically setting the sample position to the position giving the detected maximum fluorescence intensity. Spectrofluorometer. 4. A spectrofluorimeter provided with a drive mechanism capable of controlling the position, wherein the sample surface in an axial direction parallel to a sample surface of a solid or the like to be measured and parallel to a plane formed by an excitation light side and a fluorescence side optical axis. The sample surface is sequentially moved with an arbitrary amount of movement in both axial directions parallel to the sample and in the direction of the normal to the surface formed by the excitation light side and the fluorescence side optical axis. The sample surface is scanned, the position on the sample surface, the amount of movement in the normal direction, and the intensity of the fluorescence or the scattered light of the excitation light are detected and stored. Data and data in which the amount of movement in the normal direction corresponds to the axial position parallel to the side optical axis and the axial position parallel to the sample surface and perpendicular to the plane formed by the excitation light side and the fluorescence side optical axis are represented by 3 A spectrofluorimeter that outputs a figure that is displayed in three dimensions. 5. A spectrofluorimeter provided with a drive mechanism capable of controlling the position, wherein the sample surface in an axial direction parallel to a sample surface of a solid or the like to be measured and parallel to a plane formed by an excitation light side and a fluorescence side optical axis. The sample surface is sequentially moved with an arbitrary amount of movement in both axial directions parallel to the sample and in the direction of the normal to the surface formed by the excitation light side and the fluorescence side optical axis. Scanning the sample surface, detecting and storing the position on the sample surface, the amount of movement in the normal direction, and the intensity of the fluorescence intensity or the scattered light of the excitation light. Three-dimensional display of data and data with the maximum fluorescence intensity corresponding to the axial position parallel to the fluorescence side optical axis and the axial position parallel to the sample surface and perpendicular to the plane formed by the excitation light side and the fluorescence side optical axis A spectrofluorimeter, which outputs a plotted image.