JPH0572480A - Method for observing energy transfer and microscope device for the same - Google Patents

Abstract

PURPOSE:To observe the inter-molecular distance of a body to be inspected and the distribution state of molecules. CONSTITUTION:Pulsating laser light is converged on the body 5 to be inspected, fluorescent light from an extremely small point nearby the convergence point of the laser light on the body 5 to be inspected is received to detect the time base variation of the fluorescent light, and the energy transfer is observed according to the time base variation characteristic of the fluorescent light. The device for the observation has a scanning means 11 for moving the laser irradiation point and a probe relatively and an arithmetic means which finds the time base variation characteristic of the output signal of a detecting means 7.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microscope, and more particularly to a method of energy transfer observation for observing the distribution of substances at a molecular level, and a microscope therefor.

[0002]

2. Description of the Related Art In a conventional fluorescence microscope, the distribution of a fluorescent substance contained in an object to be inspected can be known by irradiating the object to be inspected with excitation light and detecting fluorescence generated in the object to be inspected. It is possible.

[0003]

However, the information obtained by the conventional fluorescence microscope is only the distribution state of a certain substance in the object to be inspected. It was at best to detect the concentration of the substance. An object of the present invention is to provide a method capable of observing an intermolecular distance and a distribution state of molecules by observing intermolecular transfer of excitation energy, and a laser fluorescence microscope suitable therefor.

[0004]

According to the energy transfer observation method of the present invention, a pulsed laser beam is focused on an object to be inspected, and a minute amount near the converging position of the laser beam on the object to be inspected. The fluorescence from a point is received, and the time change characteristic of the fluorescence is detected to obtain the time change characteristic of the fluorescence, and the energy transfer is measured based on this.

Further, the microscope apparatus according to the present invention comprises a laser light source means for supplying pulsed laser light, and a condensing optical system for condensing the laser light from the laser light source means onto an object to be inspected. A detection means including a probe for receiving fluorescence from a minute point near the laser irradiation point on the object to be inspected, a scanning means for relatively moving the laser irradiation point and the probe, And a calculation means for obtaining the time change characteristic of the output signal from the detection means.

[0006]

In general, when a molecule is excited, the excited molecule interacts with another molecule and propagates the excitation energy to the other molecule. The efficiency of the energy transfer is greatly affected by the distance between molecules in addition to the constant determined by the molecule. Moreover, since energy transfer does not occur only in one set of molecules but propagates among a plurality of molecules, the state of energy transfer between these molecules appears in the parameters of the time decay curve of fluorescence intensity. .. For example, the fluorescence intensity I (t) is expressed as I (t) = exp {-t / τ-γ (t / τ) ^{d / 6} }, where t is time, τ is fluorescence lifetime, and γ is Energy transfer efficiency, d represents dimension.

Therefore, by obtaining the time decay characteristic of the fluorescence intensity at a minute point and analyzing this characteristic, it becomes possible to observe the distance between molecules and the distribution state of molecules.

[0008]

EXAMPLES The present invention will be described below based on examples.
FIG. 1 is a schematic configuration diagram showing a specific example of a laser fluorescence microscope suitable for observing energy transfer according to the present invention.
The light from the light source means 1 composed of a pulse laser such as a mode-locked argon ion laser-excited dye laser is transmitted through the semitransparent mirror 2.
Is reflected by the objective lens 4 through the relay lens 3 and the scanner 11 as the scanning means to be focused on a minute point on the object 5. Molecules on minute points of the object to be inspected 5 are excited by the irradiated laser light. This excitation energy causes the molecule to emit fluorescence, and the excitation energy propagates to the adjacent molecule to emit fluorescence from this molecule. The fluorescence emitted from the test object 5 is received by the probe 6 and detected by the fluorescence detecting means 7. This probe 6
Is an optical fiber for a so-called near field microscope (NFM), and is suitable for detecting light from a minute point. The fluorescent light receiving means 7 photoelectrically converts the light from the probe 6 to emit a signal. On the other hand, the pulsed laser light transmitted through the semi-transmissive mirror 2 is incident on the laser light detection means 9 and a signal indicating that the excitation light pulse has been emitted is given as the single photon counting means 8
Sent to. Then, the single photon counting means 8 counts the signal from the fluorescence detecting means 7 to measure the time change of the number of fluorescence photons, and the analyzer 10 obtains the time change characteristic of the fluorescence.

In this embodiment, as the scanning means for moving the laser irradiation point and the probe 6 relative to each other, the scanner 11 for moving the condensed light by the objective lens 4 on the object 5 to be inspected is used. ing. Instead of such a scanning means, it is possible to move the probe 6 relative to the condensing optical system including the objective lens 4. Further, as the probe 6, any probe capable of receiving light from a minute point may be used, and a minute lens may be used.

Using a laser fluorescence microscope having such a configuration, with respect to a substance in which oxazine was dispersed in polymethylmethacrylic acid as a test substance, the probe 6 measured the time change of the fluorescence intensity at each minute point and measured the time. Find the decay curve. In this case, the scanning means can perform measurement by changing the distance from the point where the excitation light is collected, that is, the point where the excitation light is excited to the minute point where fluorescence detection is performed by the probe 7, and the number of fluorescent photons at any point can be measured. It is possible to determine the time decay.

FIG. 2 is an output example in which the time change of fluorescence at an excited point (energy donor) is recorded. FIG. 3 shows an example in which the output thus obtained is obtained as a time-varying characteristic curve. Curves a and b shown in FIG. 3 are examples of time decay curves of fluorescence intensity, and by analyzing this curve, the efficiency of energy transfer due to the intermolecular distance, the molecular concentration, etc. can be known. From the comparison of such attenuation curves,
It becomes clear that the intermolecular distance is smaller in the case of the attenuation characteristic of the curve a than in the case of the attenuation characteristic of the curve b.

Further, FIG. 4 shows an example of a time decay curve of fluorescence at a minute point (energy acceptor) near the excitation point. By analyzing this curve, for example, a time decay curve of the fluorescence intensity such as the curves c and d shown in FIG. 5 can be obtained. From these comparisons, the degree of attenuation is smaller in the case of the curve c than in the case of the curve d. From this, it can be seen that the case of the curve c is smaller than that of the curve d. It is presumed that there is a large amount of energy transfer from the observed to the minute point (acceptor), that is, the energy transfer was performed through many molecules.

As described above, by analyzing the time decay curve of the fluorescence intensity at the donor of the excitation energy and the time decay curve of the fluorescence intensity at the acceptor of the excitation energy around the donor, it is possible to determine the distance between molecules and the density. We were able to obtain information on the distribution of substances at the molecular level.

[0014]

As described above, according to the present invention, it is possible to measure the distance and distribution state of molecules at the molecular level in the object to be inspected, and obtain information that cannot be obtained by the conventional general microscope. It becomes possible.

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram of a laser fluorescence microscope according to the present invention.

FIG. 2 is an output example of changes in fluorescence intensity over time for a donor.

FIG. 3 is an example of a fluorescence intensity time decay curve for a donor.

FIG. 4 is an output example of a change in fluorescence intensity with time for an acceptor.

FIG. 5: Example of fluorescence intensity time decay curve for acceptor.

[Explanation of symbols for main parts]

 DESCRIPTION OF SYMBOLS 1 ... Pulse light source means 3 ... Relay lens 4 ... Objective lens 5 ... Test object 6 ... Probe 7 ... Fluorescence detection means 8 ... Single photon counting means 11 ... Scanner (scanning means)

Claims (2)

[Claims] 1. A pulsed laser beam is condensed on an object to be inspected, fluorescence from a minute point in the vicinity of the condensing position of the laser beam on the object to be inspected is received, and time change of the fluorescence is detected. An energy transfer observation method, which comprises detecting and measuring energy transfer based on a time-varying characteristic of the fluorescence. 2. A laser light source means for supplying pulsed laser light, a condensing optical system for condensing the laser light from the laser light source means onto an object to be inspected, and an object to be inspected. Detection means including a probe for receiving fluorescence from a minute point near the laser irradiation point, scanning means for relatively moving the laser irradiation point and the probe, and an output signal from the detection means. And a calculation means for obtaining the time change characteristic of the microscope apparatus.