JP2003254963A - Instrument for dynamically measuring biological molecule - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a device capable of measuring a biological specimen by associating its optical characteristics, dynamic characteristics, and biochemical characteristics, with each other. <P>SOLUTION: A measuring instrument 100 includes an inverted microscope 120. The microscope 120 has a first XY stage 132 and a second XY stage 142 for moving a specimen 110, an observational optical system including an objective lens 152, and a lighting optical system 210 including a light source 212 and a condenser lens 216. The instrument 100 has an optical system for observing forward scattering, which includes a light quantity detector 236. The instrument 100 has an optical system for observing fluorescence or backward scattering, which includes an excitation light source 312, a mirror scanner 318, and a light quantity detector 330. The instrument 100 has a probe scanning unit 410, having a cantilever 412 and a displacement detecting system therefor. The detecting system includes a light source 422, a light receiving element 430, and a displacement detector 440. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for measuring optical properties, mechanical properties and biochemical properties of a sample of a cell population adhered to a specific substrate, a blood cell or a biological specimen of a biomolecule. Regarding

[0002]

Japanese Patent No. 3090679 discloses optical data characterizing cell populations as a method for measuring the optical properties of biological specimens. The method comprises scanning a cell population with a laser beam to produce a digital data sample, wherein the different samples represent different locations within the cell population, and storing the digital data. Locating cells within the population, defining a neighborhood of digital data samples, including digital data samples corresponding to the located cells, and for each digital data sample in the neighborhood, before or after. Estimating a background level based on stored digital data from corresponding scan positions in the neighborhood, and using each of the digital data samples corresponding to the neighborhood using the estimated background level. And the step of And a step of generating an optical data taking the sum of the corrected digital data samples of the serial,
The cell population is on a movable surface, the scanning is performed in synchronism with a step movement of the movable surface, and a time substantially corresponding to a time when digital data is acquired is recorded and stored as synchronous digital time data, Digital position data is generated from which the position corresponding to the sample can be obtained, so that each sample has a corresponding digital time point and position.

The patent also discloses a device for producing optical data characterizing a cell population as a device for measuring the optical properties of a biological specimen. The device is a scanner that scans the cell population with a laser beam and a powered stage that moves the cell population at an angle to the laser scan, which produces a digital data sample from the light emitted from the cells by laser stimulation. Generating a different sample representing a different position along a scan line within the cell population, a scanner and a stage, a memory for storing the digital data, and a digital data sample in the memory corresponding to the cells in the population. Means for positioning, means for defining a neighborhood around said positioned cell, background level for each digital data sample in said neighborhood, stored digital data corresponding to scan positions in previous or subsequent neighborhoods. A means to estimate based on and to generate optical data ,
Means for correcting each of the digital data samples corresponding to the neighborhood using the estimated neighborhood background level of the foregoing.

Further, Japanese Patent Laid-Open No. 322552/1993 discloses a probe scanning microscope as an apparatus for measuring mechanical characteristics and biochemical characteristics of a biological specimen. This device brings the probe close to the sample to a very small distance, detects a physical quantity such as an attractive force acting between the probe and the sample, and scans the probe or the sample to determine the surface shape and surface of the sample. In a scanning probe microscope that measures physical properties,
A peak detector for detecting the maximum value of the attractive force acting between the probe and the sample, and x for scanning the probe or the sample
-Adsorption force image storage device that measures and images the adsorption force distribution from the scanning signal in the y direction and the output of the peak detection device, measures the force curve at each measurement point, and stores and images the adsorption force. By doing so, the suction force distribution is measured.

[0005]

The above-mentioned devices are capable of measuring optical properties, mechanical properties and biochemical properties, but they are separate devices. It does not make sense to use different instruments to measure biological specimens over time.

It is an object of the present invention to provide a device capable of associating and measuring optical properties, mechanical properties and biochemical properties of a biological specimen.

[0007]

According to the present invention, in a device for acquiring characteristic information of a sample, the position of the sample is detected in advance based on the optical characteristic and the detected optical characteristic is set within a certain range. It is characterized in that the position to which it belongs is automatically detected and the mechanical measurement is performed at the detected position.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the drawings.

The present embodiment is an apparatus for measuring optical characteristics, mechanical characteristics and biochemical characteristics of a biological specimen.

In FIG. 1, a measuring device 100 is a sample 1
It has an inverted microscope 120 for optically observing 10. The microscope 120 does not necessarily have to be an inverted type, as long as each functional element described later is arranged in a proper place.
It may be upright.

The sample 110 may be a tissue or cell specimen, a slide on which biomolecules such as DNA or protein dropped in a specific pattern are placed, or a culture container in which cells are attached to the bottom surface and the top surface can be opened.

The cells of the sample may be changed in light scattering property by utilizing an antibody reaction or the like or may be stained with a fluorescent substance depending on the purpose. However, the scattering or fluorescence of each cell is
It is desirable to relate to either specific cell components such as DNA or proteins, or the amount of fluorescent antibody that binds only to specific cell antigens.

The microscope 120 has a stage for moving the sample 110. The stage is the first XY
It has a stage 132 and a second XY stage 142. The first XY stage 132 is a piezoelectric controlled XY
The stage is driven by the XY piezoelectric body driving circuit 134. The second XY stage 142 is a step motor-controlled XY stage. Second XY stage 142
Has an X-direction step motor 144 and a Y-direction step motor 146, and is controlled by the XY stage control circuit 148.

The microscope 120 has an observation optical system for optically observing the sample 110. The observation optical system
An objective revolver 150, a plurality of objective lenses 152 attached to the objective revolver, a filter 162,
Mirror 166 for bending the optical path, and lower housing 16
4 and a port 168 provided in the No. 4 port. Any one of the objective lenses 152 is arranged on the optical path below the sample 110 by the objective revolver 150. Port 1
A CCD camera 172 is attached to 68, for example. An eyepiece tube or the like may be attached to the port 168. Focusing is performed on the microscope 120 manually or by moving the objective revolver 150 up and down by a motor.

The microscope 120 further has an illumination optical system 210 for illuminating the sample. Illumination optical system 21
Reference numeral 0 has a light source 212, a collector lens 214, and a condenser lens 216. Illumination optical system 210
Further has a shutter 222 and a phase difference ring opening 224. These are appropriately arranged on the optical path according to the spectroscopic method.

The measuring device 100 further has an optical system for observing forward scattering. This optical system is based on a dichroic mirror 232 arranged on the optical path of the illumination optical system 210, a photodetector 234 that outputs an electric signal according to the intensity of received light, and a signal from the photodetector 234. And a light amount detector 236 for calculating the light amount.

The measuring apparatus 100 further has a fluorescence observation optical system for observing fluorescence or backscattering.
The fluorescence observation optical system includes an excitation light source 3 for generating excitation light.
Have twelve. The excitation light source 312 is, for example, a solid laser such as a helium neon laser, an argon ion laser, or a helium cadmium laser, or a super luminescent diode. Excitation light source 312
May have two light sources depending on the application.

The fluorescence observation optical system further includes a dichroic mirror 314 for selectively extracting fluorescence, a lens 316, a mirror scanner 318 for scanning a beam of excitation light, a lens 322, and an external illumination field diaphragm. Three
24, a filter 326, and a dichroic mirror 328 arranged on the optical path of the observation optical system described above. These are arranged along the optical path of the excitation light generated by the excitation light source 312.

The dichroic mirror 314 allows laser light of almost all wavelengths to pass therethrough, while selectively reflecting the fluorescence generated in the sample 110. The mirror scanner 318 is driven by the laser scan circuit 320.

The fluorescence observation optical system also includes the objective lens 152 for optical observation described above. That is, the fluorescence observation optical system shares the objective lens 152 with the observation optical system described above. The fluorescence observation optical system and the optical path of the observation optical system are coupled by the dichroic mirror 328 only between this and the sample 110. The dichroic mirror 328 selectively reflects excitation light and fluorescence.

The fluorescence observation optical system further has a light amount detector 330 for detecting the fluorescence reflected by the dichroic mirror 314. The light amount detector 330 includes a mirror 332 and a filter 334 as shown in FIG.
And a first photomultiplier 336. The light amount detector 330 further includes a dichroic mirror 342, a filter 344, and a second photomultiplier 346. Instead of photomultiplier, photodiode, photomultiplier tube, etc.
Any photoelectric conversion element capable of detecting the amount of light may be used.

The dichroic mirror 342 may be partially silver plated and may reflect or transmit based on polarization if the application requires that fluorescence depolarization be measured.

In FIG. 1, the measuring apparatus 100 further includes a probe scanning unit 410 for observing the sample 110 with a probe, and a Z stage 452 that supports the probe scanning unit 410 so as to be movable along the Z axis. Have The probe scanning unit 410 is supported above the sample 110 by the Z stage 452. The Z stage 452 is an electric body controlled Z stage, and is a probe scanning unit 41 along the Z axis.
The position of 0 can be adjusted with high precision. Further measuring device 100
Has a Z piezoelectric drive circuit 454 for driving the Z stage 452.

Although not shown in the drawing, the measuring apparatus 100 further includes a second Z stage that supports the Z stage 452 so as to be movable along the Z axis. The second Z stage is a motor controlled Z stage and has a relatively long stroke. The second Z stage is Z stage 4
The probe scanning unit 410, which moves the probe scanning unit 410 together with 52, has a cantilever 412 having a probe at its free end, and a displacement detection system for detecting the displacement of the free end of the cantilever 412.

The displacement detection system includes a light source (LD) 422 that emits a light beam, a lens 424 that narrows the light beam, and a mirror 4 that reflects the light beam toward the cantilever 412.
26, a mirror 428 for deflecting the reflected light from the cantilever 412, a light receiving element (PD) 430 for photoelectrically converting the reflected light from the cantilever 412, and a displacement of the cantilever 412 based on an electric signal from the light receiving element 430. The measuring device 100 including the displacement detector 440 further includes an operation control device 180 for controlling the entire device. Motion control device 180
Is a so-called computer, which has a built-in storage device, analog-digital converter, and the like. Motion control device 1
80 acquires information from the light amount detector 236, the light amount detector 330, and the displacement detector 440, and the XY piezoelectric body drive circuit 13
4, an XY stage control circuit 148, and a Z piezoelectric body driving circuit 4
54 and the laser scanning circuit 320 are controlled.

The operation controller 180 includes a light amount detector 236.
The analog signal is received from the light amount detector 330 and the digital signal obtained by analog-to-digital conversion is stored. It also receives a digital value that provides a limiting value from the XY stage 142 and provides a digital output value that controls the XY stage 142. It also controls the analog voltage value used to control the supply voltage to the light intensity detector 330.

The motion controller 180 includes a displacement detector 440.
From the displacement signal of the cantilever 412, the Z piezoelectric body driving circuit 454 is controlled based on the displacement signal, and the unevenness information or molecular physical property information of the sample surface is acquired and stored. Further, the positional information of the sample 110 is acquired based on the second XY stage 132, and the positional information is associated with the concave-convex information or the physical property distribution information to construct an image of the concave-convex information or the physical property distribution information.

The light beam emitted from the excitation light source 312 passes through a dichroic mirror 314 and two lenses 316 and 3 located before and after a mirror scanner 318.
An image is formed on the external illumination field stop 324 by 22.
The light beam is moved or scanned by an external illumination field stop 324 by a mirror scanner 318. The light beam that has passed through the external illumination field diaphragm 324 passes through the filter 326 and the dichroic mirror 238, and then the objective lens 152.
The light is condensed by the light to form a spot having a constant diameter or size on the surface of the sample 110.

The focal lengths of the lenses 316 and 322 and the deflection angle of the mirror scanner 318 control the spot size and scanning length of the sample 110 according to the field stop 324. The mirror scanner 318 is driven at 100 to 1000 Hz, the spot size on the sample 110 is 10 microns, and the scan length on the sample surface is 120 microns. These are nominal values and can be changed by changing the objective lens 152 arranged on the optical path by the objective revolver 150.

The second XY stage 142 preferably has a sensor capable of monitoring the amount of movement thereof. Preferably, the sensor gives a signal for indicating the origin position of the XY stage 142 and a signal for restricting the traveling of the XY stage 142 to the operation control device 180. More preferably, the sensor detects the relative movement amount of a given cell from the reference position on the sample surface, and the motion control device 180 stores it. By rearranging the cells according to the stored migration amount, it is possible to easily observe the cells again and measure the optical characteristics of the cells again.

The fluorescence generated by the sample 110 and the light back-scattered by the sample 110 go backward in the optical path of the excitation light and reach the dichroic mirror 314, whereby the light quantity detector 3 is detected.
It is selectively reflected toward 30. The energy (fluorescence or scattered light) incident on the light quantity detector 330 is divided into two by the dichroic mirror 342 as shown in FIG. The dichroic mirror 342 divides into, for example, scattered light and fluorescent light, or into two fluorescent lights having different wavelengths.

The energy reflected by the dichroic mirror 342 is filtered by the filter 344 having an appropriate band wavelength.
And reaches the photomultiplier 346, and is photoelectrically converted. The remaining energy that has passed through the dichroic mirror 342 is reflected by the mirror 332 and is filtered by the filter 3
After passing through 34, it reaches the photomultiplier 336 and is photoelectrically converted.

The light amount detector 330 calculates the light amount based on the outputs of the photomultipliers 336 and 346.
The light quantity detector 330 adjusts the signal strength of the signal corresponding to the light quantity by an internal amplifier within the input voltage range of the analog-digital converter of the operation control device 180 and sends the signal to the operation control device 180.

Returning to FIG. 1, the light scattered forward by the sample 110 is reflected by the dichroic mirror 232, reaches the photodetector 234, and is photoelectrically converted. Light intensity detector 236
Calculates the light amount based on the output of the photodetector 234.
The light intensity detector 236 adjusts the signal intensity of the signal corresponding to the light intensity with an internal amplifier within the input voltage range of the analog-digital converter of the operation control device 180, and the operation control device 1
Send to 80.

The operation controller 180 includes a light amount detector 236.
And an input signal from the light amount detector 330 are stored in synchronization with the position of the mirror scanner 318 or the first XY stage 132 or the second XY stage 142.
Synchronization depends on how the signals that control the movement of each scanner or stage are generated. Other methods are time division from the sync pulse or simultaneous recording, correspondence with the reading arrangement of the existing waveform. Of course, this sync signal may be used as a separate input signal.

On the monitor screen, which is not described by the user, the sampling rate and various test parameters, scanning area,
You can set threshold settings. The number of digitized parameters is also preset and the amplifier gain setting and the input-output relationship, ie linear or logarithmic, can be used as additional parameters.
The output and input of the mirror scanner 318, the first XY stage 132 or the second XY stage 142 are
Used to control the sample-to-laser light in the X and Y directions, or the relative movement of the cantilevers, each of these scanners being driven by a respective motion controller.

Measurement procedure In the first step, the in-plane of the sample is determined by the forward scattering distribution or fluorescence distribution on the cells in the XY plane of the sample stored in synchronization with the drive signal of the step motor of the first XY stage. Memorize the location of the cells. The information to be stored is a light amount distribution according to the irradiation area of the irradiated laser light, the time to be averaged, and the area on the sample determined according to the scanning speed. For example, the light quantity of the area corresponding to one cell Is extracted from the stored light intensity distribution. Determine the order of the extracted positions.

In the second step, the control signal of the second XY stage 142 is set so that the position where the constant light amount determined in the first step is detected is directly below the cantilever 412.

In the third step, the cantilever 412 and the sample 110 are moved to the Z stage 4 by the second Z stage driven by a step motor or the like not shown in FIG.
It is close enough to interact within the range of motion of 52.

In the fourth step, the displacement signal of the cantilever 412, which reflects the interaction between the cantilever 412 and the sample 110, is recorded in synchronization with the vertical movement of the Z stage 452.

A series of operations from the second step to the fourth step are repeated for the number of positions within the constant light amount range set in the first step.

By the above-mentioned operation, information on the interaction between a molecule such as a receptor or sugar chain existing on the surface of a cell expressing a specific protein or DNA and the probe on the cantilever is recorded. be able to.

The above-mentioned fourth step may be modified so as to be repeated at a constant area, for example, 100 × 100 points, using the first XY stage 132. This makes it possible to record interaction distribution information.

When the sample is limited to a predetermined position like a DNA chip, the first step described above is to move the second XY stage 142 to a predetermined position. , May be modified so that the light quantity measurement operation is performed only at a place where the sample is present. Thereby, the measurement time can be shortened.

Although specific embodiments of the present invention have been described with reference to the drawings, the present invention is not limited to the above-described embodiments, and various modifications can be made without departing from the scope of the invention. May be done.

For example, the first XY stage 142 may be driven by a pulse motor or a motor with an encoder instead of the step motor. Further, the driving may be performed by different means for each axis.

Regarding the method of selecting cells based on scattered light, the cells are folded back by a retractable mirror provided in the transmission illumination optical path, and led to an optical sensor capable of receiving only light other than direct light. You may identify based on a signal. The sensor may be installed with the optical axis shifted, or may have a concentric center portion with no sensitivity.

[0048]

According to the present invention, there is provided an apparatus capable of correlating and measuring optical properties, mechanical properties and biochemical properties of a biological specimen.

[Brief description of drawings]

FIG. 1 shows the overall configuration of an apparatus according to an embodiment of the present invention.

FIG. 2 shows a configuration of a light quantity detector of the fluorescence observation optical system of FIG.

[Explanation of symbols]

100 measuring device 120 microscope 132 First XY stage 142 Second XY Stage 152 Objective lens 172 CCD camera 180 Motion control device 210 Illumination optical system 212 light source 216 Condenser lens 236 Light intensity detector 312 Excitation light source 318 Mirror Scanner 330 Light intensity detector 410 Probe scanning unit 412 cantilever 422 light source 430 Light receiving element 440 displacement detector

Claims (5)

[Claims] 1. An apparatus for acquiring characteristic information of a sample, the position of the sample is previously detected based on the optical characteristic, and the position belonging to a certain range of the detected optical characteristic is automatically detected, An apparatus for mechanically measuring a biomolecule, which comprises performing a mechanical measurement at a detected position. 2. The measuring device according to claim 1, wherein the sample is a cell population. 3. The measuring device according to claim 1, wherein the sample is an aggregate of biomolecules such as DNA and protein. 4. The measuring device according to claim 1, wherein the mechanical measurement is a measurement based on a single distance dependency. 5. The measuring device according to claim 1, wherein the mechanical measurement is a measurement based on distance dependence of a large number of points in a specific area.