JPH0540095A - Micro-fluorometer - Google Patents

Abstract

PURPOSE:To perform highly reliable quantitative evaluation by simultaneously measuring the fluorescence from a sample and the absoption of a sample at the time of micro-fluorometry. CONSTITUTION:A light source 1, a microscope, a stage 13 and a fluorescence detector 4 are provided and a power meter 7 for monitoring the intensity of exciting light is arranged to the stage 13 and a window 12 for simultaneously performing fluorometry and absorptiometry is formed to the light detecting part protecting cover 11 of the power meter 7. The protecting cover 11 is removed and the intensity of the exciting light 9 applied to the light detecting part 10 is monitored. Next, the protecting cover 11 is applied and a sample 14 is arranged on the axis of the exciting light. The sample is irradiated with the exciting light and the transmitted exciting light is detected by the power meter 7 and the fluorescence from the sample is detected by the detector 4. By simultaneously monitoring the absorption and light emission of the sample, highly reliable quantitative evaluation can be performed by one measurement.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microscopic fluorescence analyzer.

[0002]

2. Description of the Related Art Identification and identification of chemical species in a minute area,
As a quantification method, evaluation using the measurement result by the microscopic fluorescence spectrum method is performed. The quantitative determination of the chemical species is evaluated by the fluorescence intensity, in which case it is necessary to monitor the excitation light intensity. Further, by examining the dependence of the fluorescence intensity on the excitation light intensity, the measurement condition that optimizes the detection efficiency is determined. On the other hand, the concentration, film thickness, etc. are evaluated by measuring the absorbance of the sample.

[0003]

In the quantification of luminescent species by fluorescence intensity, monitoring of excitation light intensity is indispensable data, and since the frequency of measurement is high, accurate monitoring by a simpler procedure is required. .. Further, when quantifying the luminescent species, it is an important parameter to monitor the transmittance of the excitation light with respect to the sample as well as the fluorescence intensity.
In view of such problems, an object of the present invention is to provide a microfluorescence measuring device capable of easily and accurately measuring the intensity of excitation light and simultaneously monitoring the emission and absorption of a sample.

[0004]

A microfluorescence measuring apparatus according to the present invention comprises a light source, a sample setting stage, a detector and a data processing control unit, and the center or a part of the sample setting stage in the microscope section. And a power meter for monitoring the excitation light intensity. Alternatively,
Further, it is characterized in that the protective cover for the light receiving portion of the power meter has a window transparent to the excitation light.

[0005]

In the present invention, the excitation light intensity can be easily and accurately monitored by integrating the excitation light intensity monitoring power meter with the sample setting stage. In addition, the reliability of the quantitative evaluation is improved by placing the sample above the light receiving portion via the protective cover and simultaneously monitoring the emission (fluorescence) and the absorption.

[0006]

Embodiments of the present invention will now be described in detail with reference to the drawings.

FIG. 1 is a block diagram of a microfluorescence measuring apparatus which is an embodiment of the present invention. FIG. 2 is a configuration diagram of an apparatus in which a stage for installing a sample of the microscopic fluorescence measuring apparatus according to the present embodiment is equipped with a power meter for exciting light intensity monitoring.

The block diagram of the microscopic fluorescence measuring apparatus shown in FIG. 1 comprises a light source 1, a microscope 2, a spectroscope 3, a fluorescence detector 4, a transmitted light detector 5, and a data processing controller 6. The light source 1 is preferably monochromatic light such as a laser, but may be white light whose wavelength is selected by a spectroscope or a bandpass filter. However, when the microscope 2 has wavelength selectivity such as a dichroic mirror, white light such as a mercury lamp may be used. It is desirable that the stage of the microscope 2 can be controlled to move in two axial directions orthogonal to each other on a plane perpendicular to the irradiation axis of the excitation light. Detector 4
Is for detecting the light dispersed by the spectroscope 3. The detector may be a multi-channel detector or a normal detector. The detector 5 is a power meter for monitoring excitation light and sample transmitted light.

In FIG. 2, the excitation light monitoring power meter 7 is built in the center of the sample setting stage 13 or a part thereof, and by moving the stage, the excitation light 9 passing through the objective lens 8 is , Is incident on the light receiving surface of the light receiving unit 10 from the vertical direction. By mounting the light receiving section protection cover 11, the unevenness of the stage is eliminated. In the absorption measurement, as the light-receiving part protective cover, one having a window 12 transparent to the excitation light attached to the excitation light irradiation region is used.

Next, the operation of the apparatus of this embodiment will be described.

First, the protective cover 11 is removed, and the position of the sample setting stage 13 is adjusted so that the excitation light 9 forms an image at the center of the light receiving portion 10. After the light source 1 is stabilized, the excitation light intensity is monitored. Next, the protective cover 11 is installed, the sample 14 is installed thereon, and the fluorescence measurement is performed. When investigating the dependence of fluorescence intensity on excitation light intensity, the fluorescence intensity at the same site can be measured by monitoring the excitation light intensity and fluorescence intensity while repeatedly attaching and detaching the protective cover with the sample installed. Can be measured easily. When simultaneously monitoring the fluorescence intensity and the absorption of the excitation light in the sample, a protective cover provided with a window transparent to the excitation light is used in the excitation light irradiation region.

When fluorescence measurement was actually carried out using this experimental system, the intensity of the irradiation light on the sample was easily and accurately monitored. Also, when measuring the excitation light intensity dependence of the fluorescence intensity, the measurement time is shortened compared to the conventional measurements,
It showed good reproducibility. Simultaneous measurement with absorption can obtain the correlation between the relative amount of the sample and the fluorescence intensity, so even if the parameter such as film thickness or concentration is unknown, highly reliable evaluation is possible with one measurement. became.

[0013]

As described above, the microfluorescence measuring apparatus of the present invention has an effect of monitoring excitation light intensity and absorbance. According to the present invention, the sample stage is integrated with the power meter for monitoring the excitation light intensity, the excitation light intensity and the absorbance of the sample can be easily and accurately monitored, and the reproducibility and reliability of quantitative measurement are improved.

[Brief description of drawings]

FIG. 1 is a block diagram for explaining a microfluorescence measuring apparatus according to an embodiment of the present invention.

FIG. 2 is a diagram for explaining a microfluorescence measuring apparatus according to an embodiment of the present invention.

[Explanation of reference numerals] 1 light source 2 microscope 3 spectroscope 4 fluorescence detector 5 transmitted light detector 6 data processing control device 7 power meter 8 objective lens 9 excitation light 10 light receiving portion 11 protective cover 12 window 13 stage 14 sample

Claims (2)

[Claims] 1. A light source, a microscope, a sample setting stage, a detector, and a data processing control unit, and a power meter for exciting light intensity monitor is mounted on the sample setting stage. Microfluorescence measuring device. 2. The microfluorescence measuring apparatus according to claim 1, wherein a protective cover for the light receiving section of the power meter is provided with a window transparent to excitation light for simultaneously measuring fluorescence and absorption of the sample. The characteristic microscopic fluorescence measurement device.