JP4448471B2 - High time resolution imaging method and apparatus, and total reflection fluorescence microscope - Google Patents

Description

  The present invention relates to a high time resolution imaging method and apparatus capable of observing one fluorescent dye molecule and the like, and a total reflection fluorescent microscope using the same.

The development of technology related to the optical microscope in recent years is remarkable, and it has now reached the stage where one protein in an aqueous solution can be studied. This development is made possible by new optical system technologies such as total reflection illumination, the development of various types of high-sensitivity cameras, and improved optical filter characteristics. Numerous experimental methods have emerged, and a new trend of “single molecule physiology” is now emerging.
For example, molecular motors and proteolytic enzymes are considered to be closely related to their functions, accompanied by dynamic structural changes due to substrate binding.
There is a need for a technique that makes it possible to visualize the structural change that occurs in such a single biomolecule under a microscope while remaining alive at the molecular level. In order to move this big flow to the next new step, an innovative method based on a new viewpoint is required.

One technique for observing a single molecule of protein is to specifically label the protein with a fluorescent dye and capture the signal from that single fluorescent dye molecule.
A fluorescence microscope uses an optical system that emits excitation light to illuminate a fluorescent dye, using a dye that emits light of a wavelength longer than the wavelength of the light when it hits a specific wavelength. The structure which combined the optical microscope which observes the performed fluorescence is provided.
When a fluorescent dye-bonded reagent is bound to the intracellular structure to be observed and the fluorescent dye molecule is irradiated with light of a predetermined wavelength, the target intracellular structure emits fluorescence against a dark background.

The number of fluorescent dye molecules observable with a general fluorescent microscope is several tens or more, and one fluorescent dye molecule cannot be identified.
This is because noise, that is, the optical signal intensity from the surroundings is larger than the optical signal intensity obtained from one fluorescent dye molecule.
In contrast, improvements have been made to improve performance, and a fluorescence microscope has been developed that can visualize one fluorescent dye molecule by improving the properties of the filter and the quality of the objective lens.

In order to observe one fluorescent dye molecule, the fact that the fluorescent dye molecule emits fluorescence by illumination with an evanescent field is used.
Specifically, non-propagating light generated in the vicinity of the boundary surface by irradiating the boundary surface between the aqueous solution containing the target sample and the glass with laser light (total reflection illumination) at an angle greater than the total reflection angle from the glass side By illuminating the target sample with an evanescent field, the fluorescent dye molecules generate fluorescence.

 The evanescent field attenuates exponentially with respect to a direction perpendicular to the boundary surface, and the degree of attenuation depends on the refractive index and the incident angle of the laser beam. Therefore, since the evanescent field illuminates only a local region having a depth of about 150 nm in the aqueous solution from the boundary surface, the total reflection illumination has an advantage that the background light is extremely less than the illumination by the normal light. is there.

Further, even under conditions where a large number of fluorescent dye molecules (concentration ˜50 × 10 ⁻⁹ mol / liter) are present in the sample aqueous solution, the probability that fluorescent dye molecules exist on the aqueous solution side near the boundary surface is small. There is little fluorescence emitted from other than one target fluorescent dye molecule immobilized on the top. Therefore, since the noise due to the fluorescence of the background light and other fluorescent dye molecules is extremely small, the fluorescence from one desired target fluorescent dye molecule can be observed.

In single-molecule observation with total reflection illumination, for example, a protein targeted by a fluorescent dye, DNA, or a biomolecule such as ATP as a substrate is bound to a glass surface, and each molecule is observed as an independent bright spot. .
When exciting a fluorescent dye molecule, it is necessary that the vibration plane of the dye molecule and the polarization direction of the excitation light match.

However, conventional total reflection illumination has a problem that molecules whose vibration planes of dye molecules coincide with the polarization direction of excitation light are bright and can be observed, but molecules that do not match are dark and cannot be observed.
In contrast, the present inventor has produced and observed a total reflection fluorescence microscope in order to detect the structural change of a specific part of one biomolecule in real time.

The “total reflection fluorescence microscope” of Patent Document 1 by the present inventor relates to the basic concept and optical system of the technology, and has a configuration of a total reflection fluorescence microscope capable of observing a dye molecule whose vibration surface is in an arbitrary direction. Disclosure.
Patent 3577514

Patent Document 2 “Total Reflection Fluorescence Microscope and Illumination Optical System” by the present inventor discloses a total reflection fluorescence microscope capable of observing the target dye molecule regardless of the direction of the vibration moment of the sample bonded to the fluorescent dye molecule. is doing.
JP 2004-138735 A

In order to image a weak signal from one molecule as a two-dimensional image, a high-sensitivity camera such as an image intensifier or a cooled CCD is used.
Since these cameras output video signals or slower digital signals, the time resolution of the data cannot exceed 100 to 30 milliseconds (video time resolution). This upper limit of time resolution is an obstacle to the progress of single-molecule research.

  Therefore, the present invention provides an imaging method and apparatus that enables observation of a single fluorescent dye molecule with high temporal resolution, such as a millisecond order greater than the temporal resolution of video, and a total reflection type using the imaging method and apparatus. It is an object to provide a fluorescence microscope.

  In order to solve the above-described problems, the high time resolution imaging method of the present invention irradiates a fluorescent dye molecule with a laser beam and receives a bright spot signal from the fluorescent dye molecule, thereby binding to the fluorescent dye molecule or the fluorescent dye molecule. In the device for observing the tissue, the irradiation laser beam is converted into linearly polarized light by the polarization conversion member, the deflection member is rotated, the traveling direction of the bright spot signal from the fluorescent dye molecule is changed, and the rotation of the deflection member The time resolution is increased by increasing the amount of information received in one imaging frame by two-dimensionally scanning the bright spot signal from the fluorescent dye molecule on the image receiving surface.

  The high time resolution imaging apparatus of the present invention is an apparatus for observing a fluorescent dye molecule or a tissue bonded thereto by irradiating the fluorescent dye molecule with laser light and receiving a bright spot signal from the fluorescent dye molecule. , A polarization conversion member that converts the laser beam to be irradiated into linearly polarized light, and a deflecting member that is rotatably provided and changes a traveling direction of the bright spot signal from the fluorescent dye molecule, and by rotating the deflecting member, The time resolution is increased by increasing the amount of information received by one imaging frame by two-dimensionally scanning the bright spot signal from the fluorescent dye molecule on the image receiving surface.

  Here, the polarization conversion member may be provided so as to be rotatable about the optical axis of the laser beam to be irradiated and rotated in synchronization with the deflection member.

  Moreover, you may comprise a polarization converting member with a quarter wavelength plate.

 The deflecting member may be composed of a prism.

 A CCD image sensor for multiplying secondary electrons by causing electrons generated on the photocathode at the time of light reception to collide with silicon behind the element may be provided on the image receiving surface that receives the bright spot signal from the fluorescent dye molecule.

  A total reflection fluorescence microscope may be configured by attaching a fluorescent dye molecule to the high-resolution image forming apparatus of this kind so as to generate fluorescence by illumination with an evanescent field.

According to the present invention, it is possible to image two-dimensionally on the image receiving surface by changing the traveling direction of the bright spot signal from the fluorescent dye molecule. As a result, the amount of information received in one imaging frame is increased and the time resolution can be increased. For example, single molecule observation can be achieved with a time resolution of milliseconds exceeding the time resolution of video, and the time resolution of ˜100 milliseconds in the prior art is rapidly increased by two digits.
Therefore, faster dynamic features of proteins can be imaged, which contributes to the elucidation of operating principles such as molecular motors and proteolytic enzymes.

Embodiments of the present invention will be described below with reference to the drawings.
In addition, this invention was made | formed on the extension of the said patent documents 1 and 2 by this inventor, and the content currently disclosed by patent documents 1 and 2 can be utilized suitably.
The present invention has succeeded in increasing the time resolution while taking advantage of a normal optical system and microscope / camera system. For example, a high-sensitivity camera capable of millisecond observation is expected to cost about tens of millions of yen for development. In the present invention, it is replaced with a commercially available inexpensive device.

The signal from one molecule of the fluorescent dye is one bright spot, but the image receiving surface of the camera spreads two-dimensionally. Therefore, the basic principle of the present invention is to record fast time information on one video screen by scanning a bright spot signal on the image receiving surface and analyzing the intensity distribution.
In order to implement this, the bright spot signal obtained by irradiating the fluorescent dye molecules of the sample with linearly polarized laser light is changed two-dimensionally on the image receiving surface by changing the traveling direction with a rotating deflection member. Basically, it will be scanned.

FIG. 1 is an explanatory view showing a main part of a total reflection fluorescence microscope system which is a preferred embodiment of the present invention, and FIGS. 2 and 3 are other embodiments. In the figure, the main part is schematically shown, and therefore, additional members such as a condenser lens and an objective lens are omitted.
An evanescent field is created by irradiating laser light at an angle greater than or equal to the total reflection angle from the glass (20) side to the boundary surface between the aqueous solution containing the target sample (10) having a fluorescent dye and the glass (20). For this purpose, circularly polarized laser light (30) is emitted from the laser light source.

The incident laser beam (30) is first converted into linearly polarized light by the polarization conversion member (21). A quarter wave plate or the like is used for the polarization conversion member (21).
Thereafter, the incident laser light (30) is incident on the glass (20), which is a total reflection surface, via the condenser lens (22) and the objective lens (23).

The bright spot signal (31) by fluorescence from the sample (10) is branched by the dichroic mirror (24).
Then, it reaches the image receiving surface (27) through the condenser lens (25) and the deflecting member (26).

The deflection member (26) has a function of changing the traveling direction of the bright spot signal (31), and a prism or the like is used. Instead of a prism, a mirror or an element that electrically controls the direction of light can also be used.
The deflecting member (26) is rotatably installed with the traveling direction of the bright spot signal (31) as a rotation axis, and is rotationally controlled by a driving means such as a stepping motor.

The bright spot signal (32) whose optical path is changed by the deflecting member (23) such as a prism is input to a two-dimensionally expanding image receiving surface (27) such as a camera.
Here, since the deflecting member (26) rotates with the traveling direction of the bright spot signal (31) as the rotation axis, the signal image is also imaged in an arc on the image receiving surface (27) according to the rotation.

In another embodiment shown in FIGS. 2 and 3, a deflecting member (26 ′) is provided between the polarization conversion member (21) and the condenser lens (22) in the optical path of the incident laser beam (30).
Similarly to the deflection member (26), the deflection member (26 ′) is also installed so as to be rotatable about the traveling direction of the incident laser beam (30) as a rotation axis, and is rotationally controlled by a driving means such as a stepping motor. The rotation of the deflection member (26 ′) is controlled in synchronization with the rotation of the deflection member (26).

In the present embodiment, the polarization conversion member (21) is also installed so as to be rotatable about the traveling direction of the incident laser beam (30) as a rotation axis, and is rotated by a driving means such as a stepping motor, similarly to the deflection member (26 ′). Be controlled. The rotation of the polarization conversion member (21) is also controlled in synchronization with the rotation of the deflection member (26 ′).
As the polarization conversion member (21), instead of rotating the quarter wavelength plate to change the polarization direction of the incident laser beam (30), a polarizer or an element for electrically controlling the polarization direction can be used.

Thereby, according to the rotation of the deflecting member (26 ′), the incident laser light (30) passing through the rotating member rotates the annular portion of the condenser lens (22). The incident laser beam (30) that has passed through the annular portion of the condenser lens (22) is similarly totally reflected by the glass (20) after passing through the annular portion of the objective lens (23).
2 and 3 is different in that the deflecting member (26) is disposed in front of or after the condenser lens (25) immediately before the image receiving surface (27).

FIG. 4 is an explanatory diagram showing an example of a captured image obtained on the image receiving surface (27).
In the present invention, since the bright spot signal from the fluorescent dye molecule is scanned on the image receiving surface (27) spreading in two dimensions by irradiating polarized light, the bright spot is used as a line like an oscilloscope. Can be recorded.
The signal image (11) obtained on the image receiving surface (27) is picked up as an arc corresponding to the rotation (12) of the deflection member (26).

At this time, the direction (13) of the vibration moment of the fluorescent dye molecules in the sample (10) and the direction of the vibration moment of the incident laser beam (30) are reflected in the signal image (11). By analyzing the shape and intensity distribution, the direction (13) of the vibration moment of the fluorescent dye molecule can also be obtained.
That is, in a plurality of fluorescent dye molecules in the evanescent field, the fluorescent dye molecules become bright when the polarization direction of the evanescent field coincides with the direction of the vibration moment. The direction of the vibration moment can also be determined.

 Generally, an expensive device of tens of millions of yen is required to control the direction of light and the shutter. In the present invention, the optical path is bent by an inexpensive optical element such as a prism, and the rotation of the prism is simply controlled. With a simple configuration, the same effect can be obtained. According to this method, the life and brightness of the fluorescent dye can be recorded as the length and intensity of the signal image (11), respectively, and information with high time resolution can be obtained quantitatively.

 As a result, for example, F1-ATPase, which is a rotating molecular motor, and myosin, dynein, proteasome, etc. are bound to fluorescent dyes and observed with a total reflection fluorescence microscope according to the present invention. It becomes possible to clarify the mechanism by which biological supramolecules, which are composites, operate.

 In general, in order to capture a bright spot signal from one molecule of fluorescent dye with high time resolution, it is necessary to increase the intensity of excitation light in order to increase the signal intensity, but there is an upper limit to the intensity of laser light that can be incident on a lens system. is there. For example, when an image intensifier is used, the photocathode may be rapidly deteriorated.

For this, it is preferable to provide a cooled CCD or EM-CCD image sensor on the image receiving surface (27) that receives the bright spot signal from the fluorescent dye molecule.
In the EM-CCD, the electrons accumulated in the light receiving unit are output through the electron multiplying transfer unit after the horizontal transfer unit. Multi-stage transfer by the electron multiplier transfer unit can obtain electrons more than 1000 times the charge generated at the time of light reception. On the other hand, the received charge is amplified on the back side of the device, so it is on the order of milliseconds. Even if a strong signal is applied, damage on the image receiving surface can be suppressed.
Further, since the number of electrons can be increased without increasing the light receiving pixel area, there is an advantage that the optical system can be downsized. Amplification is performed at the position that is most advantageous for S / N inside the light receiving element, so that the influence of noise caused by the readout amplifier and the subsequent amplification amplifier can be avoided, and imaging with low noise and high sensitivity can be realized. Unlike a photomultiplier tube that requires a multiplication voltage of several kV, it is driven at a low voltage, and there is an advantage that there is no resolution deterioration, afterimage, and image sticking.

  According to the present invention, it is possible to improve the time resolution by two digits or more while taking advantage of a normal inexpensive optical system, a system such as a microscope or a camera. This enables the imaging of faster dynamic properties of proteins and contributes to the real-time detection of structural changes in specific parts of biomolecules. It is widely used and very useful industrially.

Explanatory drawing which shows the principal part of the total reflection type fluorescence microscope system which is one Example of this invention Same example diagram Same example diagram Explanatory drawing which shows the example of the captured image obtained on an image receiving surface

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Target sample 11 Signal image 12 Direction of rotation of polarization member 13 Direction of vibration moment of fluorescent dye molecule 20 Glass 21 Polarization conversion member 22 Condensing lens 23 Objective lens 24 Dichroic mirror 25 Condensing lens 26, 26 ′ Deflection member 27 Image receiving surface 30 Incident laser beam 31 Bright spot signal due to fluorescence 32 Bright spot signal with changed optical path

Claims (7)

By irradiating a fluorescent dye molecule with a laser beam and receiving a bright spot signal from the fluorescent dye molecule, an apparatus for observing the fluorescent dye molecule or a tissue bonded thereto,
A polarization conversion member that converts the irradiated laser light into linearly polarized light;
A deflecting member that is rotatably provided and changes a traveling direction of a bright spot signal from a fluorescent dye molecule;
The time resolution is increased by increasing the amount of information received by one imaging frame by scanning the bright spot signal from the fluorescent dye molecule two-dimensionally on the image receiving surface by rotating the deflecting member. A high time resolution imaging device. A polarization conversion member is provided to be rotatable about the optical axis of the laser beam to be irradiated,
The high time resolution imaging method device according to claim 1, wherein the high time resolution imaging method device is rotated in synchronization with the deflection member. The high time resolution imaging apparatus according to claim 1, wherein the polarization conversion member is a ¼ wavelength plate. The high time resolution imaging apparatus according to claim 1, wherein the deflection member is a prism. The image receiving surface that receives the bright spot signal from the fluorescent dye molecule
The high-time resolution imaging apparatus according to claim 1, further comprising a CCD image pickup device that causes electrons generated on the photocathode at the time of light reception to collide with silicon at the rear of the device, and to multiply secondary electrons. The total reflection type fluorescence microscope by the high time resolution imaging method apparatus according to claim 1, wherein the fluorescent dye molecule generates fluorescence by illumination with an evanescent field. By irradiating a fluorescent dye molecule with a laser beam and receiving a bright spot signal from the fluorescent dye molecule, an apparatus for observing the fluorescent dye molecule or a tissue bonded thereto,
By the polarization conversion member, the irradiation laser light is converted into linearly polarized light,
Rotate the deflecting member to change the direction of travel of the bright spot signal from the fluorescent dye molecule,
The time resolution is increased by increasing the amount of information received by one imaging frame by scanning the bright spot signal from the fluorescent dye molecule two-dimensionally on the image receiving surface by rotating the deflecting member. A high time resolution imaging method.

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