JP2006010523A - Molecule measuring instrument - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a molecule measuring instrument capable of performing measurement such as FCS measurement or the like with respect to one or a plurality of molecules over a relatively long time. <P>SOLUTION: One or a plurality of measuring target molecules are held in a region 23 wider than a measuring region 24 for performing the measurement of FCS or the like with respect to one or a plurality of the moving target molecules by trap light due to the same theory as conventional optical tweezers. The conventional optical tweezers are used in order to certainly grasp a target micro-matter (dielectric) but, in this invention, they are used without being fixedly grasped in order to be held so as not to come out of a region wide to a certain degree while permitting the free movement of the target micro-matter. For example, when FCS is measured, a holding place having an area of a degree not almost exerting effect on the Brownian movement of molecules is provided. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an apparatus for performing measurements such as fluorescence correlation spectroscopy (FCS) on single or plural molecules such as biomolecules.

In recent years, single-molecule measurement for measuring the behavior of a single molecule has attracted attention, and a measurement apparatus and method for that purpose have been proposed.
As in the past, when some measurement is performed on a large number of molecules, the obtained data is only obtained as statistics on the large number of molecules, and the behavior of individual molecules cannot be known. In addition, even if a change occurs in only a small part of a large number of molecules, it cannot be detected because the change is ignored in the data obtained as statistics. In contrast, single-molecule measurement can directly capture the behavior of the molecule.

  An example of such single molecule measurement is FCS (Patent Document 1). In FCS, a liquid containing a molecule with a fluorescent label is irradiated with a beam of light to detect fluorescence emitted by the fluorescent label. The value of the autocorrelation function of the molecule is calculated from the time change of the fluorescence. The obtained autocorrelation function value includes, for example, information related to the velocity of the molecule. In addition, since the speed of a molecule that undergoes Brownian motion increases as its mass decreases, the mass of the molecule and its change over time can be known. Here, the time change of the mass of the molecule occurs, for example, when the molecule is split.

  In the present application, the “fluorescent label” includes a fluorescent substance possessed by the molecule itself in addition to the fluorescent substance attached to the molecule.

  In order to measure a micro sample such as one molecule, it is necessary to handle the micro sample. As a means for handling this, optical tweezers as described in Patent Document 2 are known. Optical tweezers use a phenomenon in which a dielectric receives a force in the direction of the light gradient (this is called a gradient force). The micro object is attracted and held by the large gradient of the intensity of the light.

JP 2001-272346 A ([0002] to [0013], FIGS. 1 and 2) Japanese Patent Laid-Open No. 6-132000 ([0009] to [0010], FIG. 1, FIG. 2, FIG. 6)

When performing FCS measurement for one molecule, if the movement space of the molecule is sufficiently large relative to the FCS measurement area, the molecule will move away from the measurement area with time due to Brownian motion, and FCS measurement will no longer be performed. Can not be. Therefore, when it is desired to perform a measurement such as investigating a change in behavior while changing some environment (for example, temperature) for one molecule of interest, it is impossible with the conventional method.
In other measurements as well, there has been no appropriate holding means in the past when trying to observe molecules while moving the molecules almost freely.

  The problem to be solved by the present invention is to provide a molecule measuring apparatus capable of performing measurement such as FCS measurement for one or a plurality of molecules over a relatively long time.

The molecular measurement device according to the present invention, which has been made to solve the above problems,
a) a measurement unit for measuring a predetermined measurement region for one or more moving target molecules;
b) a trap light source for generating trap light for holding the target molecule in the trap region including the measurement region;
It is characterized by providing.

  The measurement unit may include a fluorescence excitation light source that irradiates the measurement region with light for exciting the fluorescent substance included in the target molecule, and a detection unit that detects fluorescence. One of such measurements is fluorescence correlation spectroscopy (FCS).

In the molecular measurement apparatus according to the present invention, one or a plurality of molecules to be measured are held in an area wider than the area to be measured. Conventionally, optical tweezers have been used to securely hold a target minute object (dielectric material), but in the present invention, rather than being fixedly held in such a manner, an area that is somewhat wide is used. It is used to keep the target minute object from moving out of the area while allowing it to move freely. For example, when FCS measurement is performed, a holding field having a width that does not substantially affect the Brownian motion of the molecule is provided.
Trap light for forming such a holding field can be realized by manufacturing a trap optical system that collects light in a wider area than conventional optical tweezers.

  The trapped light gives a momentum to the molecule to be measured in the traveling direction of the light (this force is called scattering force scattering force). The trapping light used in the molecular measuring apparatus according to the present invention holds the target molecule in a relatively wide range, so that the gradient force is weakened. Accordingly, the gradient force is weak also in the traveling direction of light, and depending on the conditions, the scattering force may be larger and the molecules in the holding field may be pushed out in the traveling direction of light. In such a case, it is desirable to further provide a reverse trap light source that irradiates the trap region with light in a direction opposite to the traveling direction of the trap light so as to confine molecules in the trap region.

  The molecular measurement apparatus of the present invention further includes a trap light source for capturing a molecule to be measured or a molecule not to be measured, and a movable trap light source capable of moving the capture region around the trap region. Can do. The trap light generated by the moving trap light source is desirably one that holds the target molecule in a fixed manner, like conventional optical tweezers. For example, the target molecule is introduced from outside the measurement region into the measurement region by capturing the target molecule outside the measurement region with the trapped light from the moving trap light source and moving the trapped light into the measurement region. can do. Introducing molecules into the measurement region in this way can be used, for example, when the number of molecules in the measurement region is changed during the measurement to see changes in the interaction between the molecules.

  Conversely, by moving the capture region formed by the moving trap light source around the measurement region, it is possible to prevent molecules other than the measurement purpose from entering the measurement region.

  In the molecular measurement device according to the present invention, the state of the molecular movement is maintained while holding the target molecule or molecules in a relatively wide field by the trapped light, that is, without greatly affecting the free movement of the molecule. Etc. can be observed. Therefore, for example, it is easy to perform measurement related to a molecule, such as watching a change in a motion state while changing the environmental condition of the molecule, or watching a change of a molecule over a long time.

One embodiment of a molecular measuring apparatus according to the present invention will be described with reference to FIGS. The measuring apparatus of this embodiment is for performing FCS measurement.
FIG. 1 is a schematic configuration diagram of a measuring apparatus according to the present embodiment. A trap light source 11 and a condensing optical system (a trap light source lens 12, a reflecting mirror 13, and an objective lens 18) for forming a trap region are provided inside the sample cell 10. In the sample cell 10, a molecule to be measured is placed together with a liquid serving as a dispersion medium. In addition, a fluorescent label is attached to the molecule to be measured.

Next, the positions of the fluorescence excitation light source 14, the excitation light source lens 15, the confocal pinhole 16, and the reflecting mirror 17 are set so as to irradiate the trap region with excitation light. A field irradiated with excitation light from the fluorescence excitation light source 14 becomes a measurement region. On the other hand, a confocal optical system and a detector 19 for collecting and detecting fluorescence emitted from the measurement region are set. A pinhole 20 for forming a confocal optical system is provided immediately before the detector 19. If the fluorescence excitation light source 14 is a laser and the position of the pinhole 16 is at the focal point of the laser beam, the pinhole 16 can be omitted.
As the trap light source 11, for example, a laser light source that oscillates laser light having a wavelength of about 1000 nm is used. As the fluorescence excitation light source 14, for example, a laser light source having a wavelength of 400 to 800 nm is used according to the fluorescent labeling substance.

  As shown in FIG. 2, the size of the trap region 23 formed at the condensing point of the trap light 21 can be set by the focal length and numerical aperture of the trap light source lens 12 and the objective lens 18. Similarly, for the fluorescence excitation light 22, an irradiation region is set in the trap region 23 by adjusting the focal length and numerical aperture of the excitation light source lens 15. This becomes the measurement region 24. In practice, the trap region 23 and the measurement region 24 in FIG. 2 are formed in a three-dimensional shape.

  By forming the trap region 23 and the measurement region 24 as described above, the molecules in the sample cell 10 can move almost freely in the trap region 23, and thus repeatedly enter and exit the measurement region 24 while performing Brownian motion. (Figure 3). While the molecule exists in the measurement region 24, the fluorescent label of the molecule emits fluorescence by the excitation light 22, and does not emit fluorescence while the molecule exists outside the measurement region 24. The FCS measurement is performed based on the time change of the fluorescence detected by the detector 19. The intensity of the trapping laser beam can be adjusted according to the diameter of the particle to be measured.

  In FIG. 2, the trapped light 21 travels in the direction of the arrow 31. The molecules to be measured are given momentum in this direction by the scattering force of the trapped light 21. When this force exceeds the gradient force for trapping, the molecule to be measured is pushed out of the trap region 23. Therefore, in such a case, a reverse trap light source (not shown) is provided so that trap light traveling in a direction 32 opposite to the traveling direction 31 of the trap light 21 is provided as shown in FIG. Thereby, the molecules 33 are stably held in the trap region 23.

  Furthermore, as shown in FIG. 5, it is desirable to form a moving trap light 41 that can move around the trap region 23. For example, as shown in FIG. 5 (a), the molecule 42 to be measured can be captured by the moving trap light 41, moved, and introduced into the trap region 23. Alternatively, as shown in FIG. 5 (b), the molecules 43 that are not measurement targets can be captured by the moving trap light 41, and the molecules 43 can be prevented from entering the trap region 23. Moreover, it is possible to prevent particles in the vicinity of the measurement region in advance from being trapped by this moving trap before the measurement, so that other particles can enter the measurement region.

The schematic block diagram which shows one Embodiment of the measuring apparatus of this invention. Schematic which shows the trap area | region and measurement area | region formed with the measuring apparatus of this embodiment. Schematic which shows the positional relationship of the molecule | numerator which carries out Brownian motion, a trap area | region, and a measurement area | region. Schematic which shows the state of the trap area | region vicinity when a reverse direction light is irradiated to a sample cell. Schematic which shows the state of the trap area | region vicinity for demonstrating moving trap light.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 ... Sample cell 11 ... Trap light source 12 ... Trap light source lens 13 ... Reflection mirror 14 ... Fluorescence excitation light source 15 ... Excitation light source lens 16, 20 ... Pinhole 17 ... Reflection mirror 18 ... Objective lens 19 ... Detector 21 ... Trap light 22 ... fluorescence excitation light 23 ... trap region 24 ... measurement region 41 ... moving trap light 42 ... molecule to be measured 43 ... molecule not to be measured

Claims (5)

a) a measurement unit for measuring a predetermined measurement region for one or more moving target molecules;
b) a trap light source for generating trap light for holding the target molecule in the trap region including the measurement region;
A molecular measurement apparatus comprising:   The said measurement part has a fluorescence excitation light source which irradiates the said measurement area | region with the light for carrying out the fluorescence excitation of the fluorescent substance which a target molecule has, and the detection part which detects fluorescence, The detection part characterized by the above-mentioned. Molecular measurement equipment.   The molecular measurement apparatus according to claim 2, wherein the measurement is fluorescence correlation spectroscopy (FCS).   The molecular measurement apparatus according to claim 1, further comprising a reverse trap light source that irradiates the trap region with trap light in a direction opposite to a traveling direction of the trap light.   5. A trap light source for capturing a molecule to be measured or a molecule not to be measured, further comprising a moving trap light source capable of moving the capture region around the trap region. The molecule | numerator measuring apparatus in any one of.

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