JP2907571B2 - Laser scanning fluorescence microscope - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a laser scanning fluorescent microscope capable of displaying a fluorescent image of a biological sample or the like containing various fluorescent dyes as labels for each fluorescent dye.

[0002]

2. Description of the Related Art As shown in FIG. 5, the emission spectrum of a fluorescent dye used for labeling a biological sample often has a relatively narrow emission wavelength range and a single emission peak.

FIG. 6 shows an apparatus for obtaining a fluorescence emission image of each sample labeled with such various kinds of fluorescent dyes. Epi-fluorescence microscope 1 as shown
01, the block in which the bandpass filter 102 and the dichroic beam splitter 103 are combined is exchanged according to the type of the fluorescent dye. That is, the light from the light source 105 is transmitted to the dichroic beam splitter 10.
The light is guided onto the sample 106 by 3 and the fluorescence from the sample 106 is observed by the camera 104 through the above-described block. A plurality of images obtained by the camera 104 are superimposed later.

FIG. 7 shows another apparatus for obtaining a fluorescence emission image for each fluorescent dye. In such an apparatus, by combining the bandpass filters 202a and 202b and the dichroic beam splitters 203a and 203b, the luminous flux is split according to the peak of the emission spectrum of each fluorescent dye. Each of the split light beams is converted into a fluorescent image by each of the plurality of video cameras 204a and 204b. Thereby, a fluorescent image can be simultaneously taken for each type of fluorescent dye, and if these are superimposed, it is possible to know which part of the sample is labeled with the fluorescent dye.

[0005]

However, in the apparatus as shown in FIG. 6, the emission luminance of a fluorescent image is generally low, so that it takes a relatively long time (several tens of seconds to take a single image). (Several minutes) (SCIENC, VOL. 230, DEC 1985, pp1401)
-1403 etc.). Therefore, when the same number of images of a sample that fluctuates over time are taken as many as the types of fluorescent dyes, the image fluctuates due to deformation of the sample at the time of imaging, and there is a problem that these cannot be completely superimposed. Was.

Further, in the apparatus as shown in FIG. 7, the same number of video cameras as the types of fluorescent dyes are required, and it is difficult to completely optically match the positions, magnifications, etc. between the video cameras. In particular, when an image image intensifier is interposed to increase the sensitivity of a video camera, perfect image matching cannot be expected because non-linear distortion of each image image intensifier is superimposed. There was a problem.

Further, as a problem common to the apparatuses shown in FIGS. 6 and 7, in order to completely separate multiple fluorescence spectra, it is necessary to select only wavelengths at which the fluorescence spectra do not overlap, and to take an image. The optical path must have a characteristic of passing only a very narrow wavelength range, and there is a problem that the utilization efficiency of the light beam is low.

Accordingly, the present invention provides a laser scanning fluorescence microscope capable of separating and identifying emission images from a sample containing various types of fluorescent dyes with high S / N ratio without time lag for each fluorescent dye. The purpose is to:

[0009]

In order to solve the above-mentioned problems, a laser scanning fluorescence microscope according to the present invention comprises scanning means for scanning a condensed light spot on a sample, and detecting fluorescence generated from the sample. And a control unit for reading out the electric signal from the detection unit in synchronization with the scanning unit. Here, the light detecting means includes a first photodetector whose total spectral sensitivity characteristic monotonically increases in a predetermined spectral wavelength range, and a second photodetector whose total spectral sensitivity characteristic monotonously decreases in the predetermined spectral wavelength range. And an optical means for individually guiding the fluorescence from the sample to the first and second photodetectors. Further, the control means includes a discriminating means for discriminating which kind of fluorescent dye the fluorescence from the sample is based on the intensity ratio of the electric signal from the first and second photodetectors. The overall spectral sensitivity characteristic refers to the overall spectral sensitivity characteristic of a system in which a photodetector and optical means are combined, such as the spectral sensitivity characteristic of a photodetector, and a demultiplexer placed in front of each light detection part. It is determined by a combination of other spectral demultiplexing characteristics, filters and other spectral transmittance characteristics, and the like.

[0010]

According to the above laser scanning fluorescence microscope, the total spectral sensitivity characteristic of the first photodetector monotonically increases in a predetermined spectral wavelength range, and the total spectral sensitivity characteristic of the second photodetector is the same spectral wavelength. Monotonically decreases in the range. Therefore, the center wavelength of light emission can be specified based on the intensity ratio of the electric signals from the first and second photodetectors, and the emission spectrum of each of a plurality of types of fluorescent dyes is relatively narrow. Assuming that the fluorescent dye has a single emission peak, the type of these fluorescent dyes can be specified or determined.
If such determination of the fluorescent dye is performed in synchronization with the scanning means, a fluorescent image corresponding to the intensity of each type of fluorescent dye can be obtained without a time lag.

[0011]

An embodiment of the present invention will be described below with reference to the accompanying drawings.

FIG. 1 is a schematic diagram showing an optical system of a laser scanning fluorescence microscope of an embodiment. As shown, a laser beam for excitation from a continuous wave laser light source 1 travels straight through an imaging lens 7, a pinhole 8, a collimator 9 and a dichroic mirror 10, and a galvanometer mirror provided in the first scanner 2. Incident on. This first scanner 2
The laser beam deflected by the above enters the galvanometer mirror provided in the second scanner 3 via the relay lenses 34 and 35, and is deflected again. On the emission side of the second scanner 3, the objective lens 15 is arranged via an eyepiece 36, and these form a laser spot light on the sample 6. The laser spot light is scanned on the sample in the X and Y directions by the first and second scanners 2 and 3 as scanning means, respectively.

The fluorescence emitted from the sample 6 is sequentially deflected by the second scanner 3 and the first scanner 2, and then enters the dichroic mirror 10. Since the dichroic mirror 10 reflects only a light beam in a wavelength region longer than the wavelength of the laser light beam, fluorescence having a wavelength longer than the wavelength of the laser light beam is reflected by the dichroic mirror 10,
The light enters a dichroic mirror 13 via an imaging lens 11 and a pinhole 12. The fluorescence split by the dichroic mirror 13 is divided into filters 14 and 1 respectively.
6. First and second photomultipliers etc.
And is converted into an electric signal at the same time. The overall spectral sensitivity characteristics of the optical path on the first photodetector 15 side include not only the spectral sensitivity characteristics of the first photodetector 15 itself, but also the spectral transmittance characteristics of the filter 14 and the spectral demultiplexing characteristics of the dichroic mirror 13. And so on. The overall spectral sensitivity characteristic of the optical path on the second photodetector 17 side is also given by a combination of the spectral characteristics of the photodetector 17, the filter 16, the dichroic mirror 13, and the like. Although described in detail below, the overall spectral sensitivity characteristic of the optical path on the first photodetector 15 side monotonically increases with wavelength in a predetermined spectral wavelength range, and the overall spectral sensitivity characteristic of the optical path on the second photodetector 17 side. The sensitivity characteristic monotonically decreases with wavelength in the same wavelength region.

FIG. 2 is a schematic diagram showing a control system of the laser scanning fluorescence microscope of the embodiment. The electric signals from the photodetectors 15 and 17 are converted into digital signals by the A / D converters 21 and 23, and then synchronized with the drive signals of the scanners 2 and 3, and the same addresses of the pair of image memories 25 and 27 are synchronized. Are sequentially written to the pixels and stored as image signals. The control analyzer 30 as a discriminating unit discriminates the type of the fluorescent dye for each pixel based on a pair of data stored in each of the image memories 25 and 27. Data relating to the type of fluorescent dye obtained for each pixel is compiled into an image and displayed on the display 32 as, for example, a concentration distribution of a specific type of fluorescent dye.

The operation of the laser scanning fluorescence microscope of the embodiment will be described below. First and second scanners 2, 3
Is based on the drive signal from the control analyzer 30.
The laser spot light irradiated above is scanned. From the sample 6, fluorescence is generated in accordance with the distribution of the fluorescent dye used for preparing the sample. This fluorescence is transmitted to the first and second photodetectors 1.
5 and 17 are detected simultaneously, but the output ratio of each of the photodetectors 15 and 17 differs depending on the position of the peak wavelength.

FIG. 3 compares the total spectral sensitivity characteristic of the optical path of each of the photodetectors 15 and 17 with the emission spectrum of the fluorescent dye. The overall spectral sensitivity characteristic of the first photodetector 15 covers an appropriate spectral wavelength range λ _{1 to} λ ₂ ,
And the sensitivity increases in a minor manner with the wavelength (see FIG. 3).
(A) chain line). On the other hand, the overall spectral sensitivity characteristic of the second photodetector 17 covers the spectral wavelength range λ _{1 to} λ ₂ , and its sensitivity decreases in a minor tone with wavelength (solid line in FIG. 3A). As the fluorescent dye, a dye whose emission spectrum distribution is known in advance is used. That is, three kinds of fluorescent dyes that cover an appropriate region within the above-mentioned spectral wavelength range λ _{1 to} λ ₂ and have different peak wavelengths are used (FIG. 3B).

When a fluorescent dye as shown in FIG. 3B is used for preparing a sample, the output ratio of each of the photodetectors 15 and 17 having the characteristics shown in FIG. It shows an almost constant value for each type and hardly depends on the absolute value of the fluorescence intensity. Specifically, the output ratios obtained by the photodetectors 15 and 17 for the three fluorescent dyes shown in FIG. 3B are about 1: 4 for the first dye and about 1: 4 for the second dye. 1 and about 4: 1 for the third dye.

Therefore, the two pixel memories 25 and 27
By taking the ratio of the image signals stored in each pixel for each pixel, the emission images of a large number of fluorescent dyes can be separated and identified only by using two sets of photodetection systems. Further, since the type of the fluorescent dye and the fluorescence intensity of each pixel can be detected based on the overall spectral sensitivity characteristics, the distribution of the fluorescence intensity for each fluorescent dye can be obtained. Furthermore, if these are superimposed on each pixel, the whole image of the sample can be reproduced. The control analyzer 30 analyzes the intensity ratio of the image signal, the type of the fluorescent dye, and the like, and displays the fluorescent intensity distribution of the sample, the identification of the fluorescent dye, and the like on the display 32.

As is clear from the above description, in the apparatus of the embodiment, it is possible to separate and identify the emission images of various types of fluorescent dyes from each image signal only by dividing the optical path from the sample into two systems. Also, multi-wavelength fluorescent images of various types of fluorescent dyes can be simultaneously separated and identified without any image shift.
Furthermore, since the fluorescent light beam can be used without waste, an image having a good S / N ratio can be obtained in a short time.

The present invention is not limited to the above embodiment. By using a so-called confocal mode laser scanning fluorescence microscope using the pinhole 12 in FIG. 1, a fluorescence image of the sample can be three-dimensionally decomposed. In this case, the diameter and position of the pinhole 12 are adjusted, and the pinhole 12 is arranged so as to transmit only the center of the fluorescent light spot. Sample 6
Is moved up and down in the optical axis direction, the fluorescence distribution in the Z axis direction can be obtained. Accordingly, the fluorescent light that has been overlapped in the state observed two-dimensionally can be identified without overlapping, and the type of the fluorescent dye can be more reliably and easily identified. When only two-dimensional observation is performed, the pinhole 12 can be omitted.

Also, one of the A / D converters 21 and 23 is omitted, the input and output of the remaining A / D converters are switched, and the image signals are alternately stored at the same address in the two image memories 25 and 27. For example, the effect of the displacement due to the deformation of the sample corresponds to the scanning time for half a pixel, and does not cause a practical problem.

Further, one of the image memories 25 and 27 is omitted, and a division circuit, a discrimination circuit, an addition circuit and the like are provided between the A / D converters 21 and 23 and the remaining image memories.
A fluorescent dye identification bit may be added to the fluorescence intensity data as an image signal input to the image memory. If a pseudo color is presented according to the emission color of the identified fluorescent dye, an image that does not differ from that observed with the naked eye can be reproduced.

Further, three or more photodetectors may be prepared. When three photodetectors are provided, the overall spectral sensitivity characteristics of each photodetector in the spectral wavelength range to be detected can be as shown in FIG. By comparing three electrical signals from each photodetector, the accuracy of detecting the type of fluorescent dye can be increased.

[0024]

As described above, according to the laser scanning fluorescence microscope of the present invention, the total spectral sensitivity characteristics of the first and second photodetectors monotonically increase and decrease, respectively. The emission wavelength can be specified based on the intensity ratio of the electric signal from the detector, and further, the type of the fluorescent dye can be specified. Therefore, emission images from a sample containing various types of fluorescent dyes can be determined for each fluorescent dye at a high S / N ratio without a time lag.

[Brief description of the drawings]

FIG. 1 is a configuration diagram of an optical system of a laser scanning fluorescence microscope according to an embodiment.

FIG. 2 is a configuration diagram of a control system of the laser scanning fluorescence microscope of the embodiment.

FIG. 3 is a diagram showing the relationship between the overall spectral sensitivity characteristics of the photodetectors of FIGS. 1 and 2 and the fluorescence intensity distribution of the fluorescent dye used for preparing the sample.

FIG. 4 is a diagram showing the overall spectral sensitivity characteristics when three photodetectors are used.

FIG. 5 is a diagram showing a general fluorescence intensity distribution of a fluorescent dye.

FIG. 6 shows a conventional apparatus for individually detecting fluorescence emission images of a sample labeled with various kinds of fluorescent dyes.

FIG. 7 shows another conventional apparatus for detecting a fluorescence image.

[Explanation of symbols]

 2, 3, ... scanning means 10, 13, 14, 15, 16, 17 ... light detecting means 21, 23, 25, 27, 30 ... control means 17 ... first light detector 15 ... second light detector 13 ... Optical means 30 ... Discriminating means

Claims (4)

(57) [Claims] A scanning means for scanning a condensed light spot on a sample; a light detecting means for detecting fluorescence generated from the sample and converting the fluorescence into an electric signal; and the light detecting means in synchronization with the scanning means. Control means for reading an electrical signal from a laser scanning fluorescence microscope, wherein the light detection means,
A first photodetector having an overall spectral sensitivity characteristic monotonically increasing in a predetermined spectral wavelength region, a second photodetector having an overall spectral sensitivity characteristic monotonically decreasing in the predetermined spectral wavelength region, and a fluorescent light from the sample. Optical means for individually guiding the light to the first and second light detectors, wherein the control means controls the fluorescence from the sample based on the intensity ratio of the electric signals from the first and second light detectors. 1. A laser scanning fluorescence microscope, comprising: a discriminating means for discriminating which kind of fluorescent dye is used. 2. The scanning device according to claim 1, wherein the scanning unit includes a deflecting device for deflecting a laser beam from a light source, and an objective lens for condensing the laser beam as a light spot on a sample. Laser scanning fluorescence microscope. 3. A display for two-dimensionally displaying the distribution of the fluorescent dye in the sample for each type based on the scanning information of the light spot by the scanning unit and the information on the type of the fluorescent dye by the determining unit. The laser scanning fluorescence microscope according to claim 1, further comprising a device. 4. The photodetector includes a third photodetector having a total spectral sensitivity characteristic monotonically increasing in another spectral wavelength region adjacent to a longer wavelength side than the predetermined spectral wavelength region. 2. A laser scanning fluorescence microscope according to claim 1, wherein said one photodetector has its total spectral sensitivity characteristic monotonously decreasing in said another spectral wavelength range.