JP2005099054A - Fluorometer for micro matter - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorometer for micro matter that can stably measure fluorescence from micro matter over a long time. <P>SOLUTION: The fluorometer for micro matter, which houses a sample solution including molecular-level micro matter in a dish 16 and irradiates it with excitation light to conduct molecular level fluorometry on the bottom, has an evanescent illumination device 12, having intermitting means 24 for irradiating a micro area at the bottom of the dish 16 with excitation light more than once, and an objective lens 15 vertically movable relative to the dish 16. The objective lens 15 is focused on the inner bottom of the dish 16, to measure the fluorescence from the micro matter fixed to or suspended over the inner bottom of the dish 16 stably over a long time. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a fluorescence measuring apparatus for a minute substance that measures fluorescence of a minute substance using fluorescence by an evanescent wave.

  Conventionally, for example, for the purpose of observing one molecule such as protein or ATP (adenosine triphosphate), fluorescence observation is used to observe the fluorescence of each protein or ATP molecule by an evanescent wave.

 In this case, these proteins, ATP, and other samples are realized in such a state that the sample particles are bound to fluorescent beads and sandwiched between glass substrates or fixed on a glass substrate and distributed two-dimensionally (planarly). In this state, an evanescent wave is excited, and fluorescence observation that emphasizes the specificity of sample particles is performed using the fact that fluorescence is emitted from the sample.

  Here, the fluorescent beads to which the sample particles are bonded are of the same size as the sample particles and have a size of several to several tens of nm. Also, evanescent illumination uses so-called oozing light that is excited to the opposite side of the reflecting surface when the laser light is totally reflected inside the prism, for example, and the intensity of the evanescent field depends on the intensity of the incident light and the incident angle. Here, the fluorescence excitation capability can be obtained only in the region corresponding to the height of the sample particles from the substrate surface.

  By the way, in the fluorescence observation used in such a microscopic material microscope, the fluorescent agent has a fading color, and there is a limit to the observable time. This time is determined by the amount of the fluorescent agent, the intensity of the excitation light, and the brightness of the microscope used for observation. Particularly, if the size of the sample particle to be observed is large, it can be stained with as many fluorescent agents as possible. Therefore, the fluorescence is bright and can last for a long time.

  However, in the case where the size of the sample particle is extremely small as described above, the amount of the fluorescent agent with respect to the sample particle is extremely small, and the fluorescence of the observation target is lost in a short time due to the fading.

  In such a case, the observation target is changed to another one, and in some cases, the in-focus state is again adjusted so that the observation is continued. Under the light, the other fluorescent objects are also fading in the fluorescent agent, so that there is a problem that stable fluorescent observation cannot be obtained.

  On the other hand, for example, in the mechanism of the actomyosin system, which is one of the basic units of muscle contraction, the energy for moving the actin filament of myosin (protein molecule) is due to ATP hydrolysis, and this ATP consumption is observed. However, in this observation, when the myosin molecule is stained with cy5, the time during which one myosin molecule can be observed with fluorescence is at most about 60 seconds. Development of a method that continues to emit fluorescence for a longer time than before has been desired.

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a fluorescence measuring apparatus for a minute substance that can stably measure fluorescence from the minute substance for a long time.

According to the first aspect of the present invention, a sample solution containing a molecular level minute substance is accommodated in a predetermined container, the excitation light is irradiated to the container, and the fluorescence of the minute substance that performs the molecular level fluorescence measurement on the bottom surface. In the measurement apparatus, a light irradiation mechanism that performs excitation light irradiation a plurality of times on a minute region on the bottom surface of the container, and a drive mechanism of an objective lens that can move up and down with respect to the container,
The drive mechanism focuses the objective lens on the inner bottom surface of the container, thereby stably measuring fluorescence from a minute substance fixed or floating on the inner bottom surface of the container for a long time.

  According to a second aspect of the present invention, the light irradiation mechanism is configured to be capable of two-dimensional scanning with respect to the vicinity of the bottom surface of the container, and measures the video rate.

  As a result, according to the first aspect of the present invention, the fluorescence can be stably measured for a long time even with an observation target of about one molecule having a short fluorescence lifetime.

  In addition, according to the second aspect of the present invention, it is possible to follow a fast change and movement of the fluorescence of the sample in accordance with the measurement target.

  According to the present invention, the fluorescence can be measured stably for a long time even with an observation target of about one molecule having a short fluorescence lifetime. Further, it is possible to follow high-speed changes and movements of the fluorescence of the sample according to the measurement object.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

(First embodiment)
FIG. 1 shows an example in which the microscopic material inspection apparatus of the present invention is applied to an upright microscope.

  In the figure, 11 is a stage of an upright microscope, and this stage 11 is provided with an evanescent illumination device 12. The illuminating device 12 has a frame 13, and the frame 13 can be moved in a direction perpendicular to the paper surface by a guide 14 with respect to the stage 11.

  The frame 13 of the evanescent illumination device 12 has an upper surface flush with the surface of the stage 11 and a through hole 131 is formed in a portion facing an objective lens 15 described later.

  The dish 16 is placed on the through hole 131. The dish 16 contains an aqueous solution 17 in which sample particles such as protein and ATP are fixed or suspended on the inner bottom surface. In this case, each sample particle such as protein or ATP is labeled with a fluorescent agent such as cy3 or cy5.

  The dish 16 is held by a clenmel 34 of the stage 11 so that it can move in the horizontal direction in the figure. Instead of the dish 16, a sample particle sandwiched between a slide glass and a cover glass may be used.

  An evanescent illumination prism 18 is held at a position corresponding to the through hole 131 in the frame 13 of the illumination device 12. In this case, a matching oil 19 is interposed between the prism 18 and the dish 16. This matching oil 19 does not cause total reflection of laser light, which will be described later, incident on the prism 18, but only on the inner bottom surface of the dish 16 without generating it on the upper surface of the prism 18 or the outer bottom surface of the dish 16. Is.

  Further, a semiconductor laser oscillator 21 is provided on a support base 20 in the frame 13 of the evanescent illumination device 12. The semiconductor laser oscillator 21 oscillates a predetermined laser beam. The laser beam output from the laser oscillator 21 is deflected by a mirror 23 through a lens 22 and is incident on a prism 18. In this case, if the dish 16 is made of glass, its refractive index Nd = 1.42, and the refractive index naq = 1.33 of the aqueous solution 17, the incident angle of the laser beam applied to the bottom surface of the dish 16 via the prism 18 is θ = If the angle is 20 degrees or less, total reflection can be caused, and thereby, an evanescent field is generated near the inner bottom surface of the dish 16 to perform evanescent illumination.

  Note that a surface treatment layer 35 is formed on the portion of the evanescent illumination device 12 where the laser beam emitted from the prism 18 inside the frame 13 is irradiated so that the laser beam does not become stray light due to irregular reflection.

  The laser beam path between the lens 22 and the mirror 23 is provided with an interrupting means 24 for interrupting the laser beam incident on the prism 18. As shown in FIGS. 2 (a) and 2 (b), the intermittent means 24 includes a conductive disc disk 242 formed with a plurality of (two in the illustrated example) slits 241 at equal intervals in the circumferential direction by a shaft 244. A support base 245 is rotatably supported. In this case, the shaft 244 is supported on the support base 245 by a hub 243 having a sharpened tip so that the disc disk 242 can be smoothly rotated with little frictional resistance. Further, as shown in FIG. 2C, U-shaped electromagnets 246 and 247 are arranged on the disc disk 242 so as to sandwich the surface of the disk 242, and the disc disk 242 surface is perpendicular to these electromagnets 246 and 247. A rotational driving force for the disk 242 is obtained by an eddy current on the surface of the disk 242 generated by the transmitted magnetic flux.

  Such an intermittent means 24 has a disc disk 242 disposed in the laser beam path between the lens 22 and the mirror 23, and the prism 242 is inserted into and removed from the laser beam path as the disc disk 242 rotates. The laser beam irradiated to the total reflection surface from 18 is intermittently generated, and the evanescent field excitation on the inner bottom surface of the dish 16 is intermittently generated. In other words, the excitation intensity per unit time can be reduced by intermittently generating the evanescent excitation light on the inner bottom surface of the dish 16. Incidentally, if 90% of the time is cut by the slit 241 and the laser beam is allowed to pass through only 10% of the time, the excitation intensity becomes 1/10 compared to the continuous case.

  FIG. 3 shows a control circuit for the laser oscillator 21 and the intermittent means 24. In this case, a controller 25 is connected to the laser oscillator 21. This controller 25 has a power source 251 and a laser driver 252, and the laser driver 252 activates the laser oscillator 21 to generate laser light. In addition, the frequency modulator 26 is connected to the power source 251 of the controller 25, and the electromagnets 246 and 247 of the intermittent means 24 are connected to the frequency modulator 26.

  The frequency modulator 26 can change the output frequency according to the operation of the operation unit 27. The frequency modulator 26 can change the time change rate of the magnetic flux passing through the disk 242 according to the output frequency at this time. Thus, the rotational speed of the disk 242 due to the eddy current can be varied so that the intermittent period of the laser beam can be changed.

  Here, the rotational speed of the disk 242 is changed by frequency modulation. However, it is also possible to change the rotational speed of the disk 242 by changing the voltage (current) supplied to the electromagnets 246 and 247. .

  Returning to FIG. 1, an immersion type objective lens 15 is provided corresponding to the aqueous solution 17 of the dish 16. The objective lens 15 captures the state of fluorescence of the sample particles by the evanescent excitation light on the inner bottom surface of the dish 16 as an observation image by the transmitted illumination light supplied from the transmitted illumination light source 30 through the condenser lens 31, and further, the lens barrel 32. Through the imaging unit 33 so that the fluorescence reaction of the sample particles can be observed.

  In this case, the objective lens 15 is provided with a plurality of various magnifications in a revolver (not shown) so that a desired magnification can be selected. The objective lens 15 can be driven in the vertical direction to change the focal point so that it can be focused on the inner bottom surface of the dish 16. In order to obtain focus, the stage 11 may be driven in the vertical direction.

  In this way, the disc disk 242 having the slits 241 is provided between the laser oscillator 21 and the prism 18, and the disc disk 242 is rotated so that the total reflection surface in the prism 18 is provided. Since the emitted laser light is intermittently generated and the evanescent excitation light on the inner bottom surface of the dish 16 is intermittently generated, the intensity per unit time of the excitation light can be reduced, and thus the emission time can be reduced. Even with a short observation target of about one molecule, the fluorescence can be maintained for a long time, and the sample can be observed over a long time.

  Further, the frame 13 of the evanescent illumination device 12 can be moved in the direction perpendicular to the paper surface by the guide 14 with respect to the stage 11, and the dish 16 is held by the clenmel 34 of the stage 11 and can move in the horizontal direction in the figure. Therefore, if the total reflection position of the laser beam in the prism 18 is moved along the plane perpendicular to the paper surface including the optical axis of the objective lens 15 by the movement of the frame 13, then Can move the sample onto the optical axis of the objective lens 15 only by moving the dish 16 left and right by the Clemmel 34, and can easily position the objective lens 15 on the optical axis, which is the observation position of the sample. .

  In the above description, the disk 242 having the slit 241 is rotated. However, the slit 241 is fixed on the optical path of the laser beam, and the reflection surface of the mirror 23 is moved so that the irradiation angle changes. Good (rocking motion). In this case, the swing motion of the mirror 23 is automatically performed using the swing motion mechanism. Further, instead of the mirror 23, a polygon mirror having a plurality of mirrors arranged on the outer peripheral surface may be used, and the rotation axis of the polygon mirror may be rotated perpendicular to the paper surface so that each mirror surface is equivalent to the illustrated mirror 23. Similar effects can be obtained. Furthermore, a liquid crystal shutter can be considered as an alternative to the rotation of the disk 242 having the slit 241. In the liquid crystal shutter, since the ratio between the time for passing the laser beam and the time for blocking it can be changed, the fluorescence duration can be varied by controlling the fluorescence intensity per unit time.

(Second Embodiment)
FIG. 4 shows a schematic configuration of the second embodiment, and the same components as those in FIG.

  In this case, scanning means 40 formed by adhering a piezoelectric body 42 on an AOD (Acoustic Optical Device) 41 is provided in the laser optical path between the lens 22 and the mirror 23, and the piezoelectric body 42 is vibrated to generate ultrasonic waves. The laser light incident on the prism 18 is scanned by bending the optical path of the spot-like laser light in the ultrasonic field of the AOD 41.

  In this case, if the intensity of the ultrasonic wave by the piezoelectric body 42 can be controlled, the scanning width of the laser light can be changed, so that the intermittent period of the laser light incident on the prism 18 can be changed. Further, another piezoelectric body is bonded in a direction perpendicular to the piezoelectric body 42 on the AOD 41, ultrasonic waves are also generated from the other piezoelectric body, laser light is scanned, and scanning with the piezoelectric body 42 is performed in combination. By doing so, two-dimensional raster scanning can also be performed.

  Even in this case, since the laser light to the prism 18 can be intermittently generated and the evanescent excitation light on the inner bottom surface of the dish 16 can be generated intermittently, the excitation per unit time of the evanescent light can be shortened to reduce the fluorescence. Scarring can be reduced. In addition, by using the AOD 41, it becomes possible to scan the laser beam at a speed of about 100 times / second by two-dimensional scanning, so that it can follow high-speed changes in the fluorescence of the sample and high-speed movement. be able to.

  Therefore, even in this way, the same effect as that of the first embodiment can be expected. For example, even when an image is formed at a video rate, observation can be performed for a long time. Further, since the vibration of the AOD 40 itself is small, even if the intermittent means 40 using such an AOD 40 is arranged near the sample, the influence of the vibration on the sample can be reduced.

  As mentioned above, although embodiment was described, the following invention is also contained in this invention.

(1) The evanescent wave excitation light is intermittently passed through the slit.

  In this way, even the observation object of about one molecule with a short fluorescence lifetime can sustain the fluorescence for a long time.

(2) In (1), the slit is provided in the disk disk, and the excitation light is interrupted by rotating the disk disk.

  In this way, the excitation light can be interrupted with an inexpensive configuration.

(3) The evanescent wave excitation light is intermittently changed by changing the reflection angle of the mirror.

  In this way, the excitation light can be interrupted with an inexpensive configuration.

(4) In (3), the reflection angle of the mirror is automatically changed using a swing motion mechanism.

  In this way, the excitation light can be interrupted with an inexpensive configuration.

(5) In (3), the mirror is a polygon mirror, and the polygon mirror is driven to rotate.

  In this way, the mirror rotation mechanism can be simplified.

(6) The excitation light of the evanescent wave is interrupted by intermittently deflecting the spot light.

  In this way, even the observation object of about one molecule with a short fluorescence lifetime can sustain the fluorescence for a long time.

(7) In (6), AOD is used for deflection scanning of spot light. In this way, the influence of vibration on the sample can be reduced.

  In addition, this invention is not limited to the said embodiment, In the implementation stage, it can change variously in the range which does not change the summary.

  Furthermore, the above embodiments include inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent elements. For example, even if some constituent requirements are deleted from all the constituent requirements shown in the embodiment, the problem described in the column of the problem to be solved by the invention can be solved, and is described in the column of the effect of the invention. If the above effect is obtained, a configuration from which this configuration requirement is deleted can be extracted as an invention.

The figure which shows schematic structure of the 1st implementation of this invention. The figure which shows schematic structure of the interruption means used for 1st Embodiment. The figure which shows schematic structure of the control part used for 1st Embodiment. The figure which shows schematic structure of the 2nd Embodiment of this invention.

Explanation of symbols

11 ... Stage, 12 ... Evanescent lighting device,
13 ... Frame, 131 ... Through hole,
14 ... guide, 15 ... objective lens,
16 ... Dish, 17 ... Aqueous solution,
18 ... Prism, 19 ... Matching oil,
20 ... support stand, 21 ... semiconductor laser oscillator,
22 ... Lens, 23 ... Mirror,
24: Intermittent means, 241 ... Slit,
242 ... disc, 243 ... hub,
244 ... axis, 245 ... support base,
246, 247 ... electromagnet, 25 ... controller,
251 ... Power supply, 252 ... Laser driver,
26: Frequency modulator, 27: Operation unit,
30 ... Light source for transmitted illumination, 31 ... Condenser lens,
32 ... Lens barrel, 33 ... Imaging unit, 34 ... Clemmel,
35 ... surface treatment layer, 40 ... scanning means,
41 ... AOD, 42 ... Piezoelectric material

Claims (2)

In a fluorescence measuring apparatus for a micro substance that contains a sample solution containing a micro substance at a molecular level in a predetermined container, irradiates the container with excitation light, and performs a fluorescence measurement at a molecular level on the bottom surface.
A light irradiation mechanism that performs excitation light irradiation a plurality of times on a minute region on the bottom surface of the container;
An objective lens drive mechanism capable of moving up and down relative to the container;
The driving mechanism causes the objective lens to focus on the inner bottom surface of the container, so that the fluorescence from the minute substance fixed or floating on the inner bottom surface of the container is stably measured for a long time. Fluorescence measuring device. The apparatus for measuring fluorescence of a minute substance according to claim 1, wherein the light irradiation mechanism is configured to be capable of two-dimensional scanning with respect to the vicinity of the bottom surface of the container, and measures the video rate.

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