Optical: systems and elements – Compound lens system – Microscope
Reexamination Certificate
2000-03-17
2003-12-23
Robinson, Mark A. (Department: 2872)
Optical: systems and elements
Compound lens system
Microscope
C359S385000, C250S458100, C250S459100
Reexamination Certificate
active
06667830
ABSTRACT:
TECHNICAL FIELD
The invention of this application relates to a microscope system. More particularly, the invention of this application relates to a novel microscope system of high performance and function, which is able to achieve a high quality image of a high spatial resolution by illuminating a dyed specimen with lights of plural wavelengths.
BACKGROUND ART
In the prior art, there have been developed various types of optical microscopes, and their performance has been enhanced according to the development in the peripheral technique including the laser technique and the electronic graphic technique. As one of these high performance optical microscopes, there has been proposed (in Japanese Patent Application No. 6-329165) a microscope which is able, by using a double resonance absorption process induced by illuminating a specimen with lights of plural wavelengths, not only to control the contrast of an image to be obtained but also to perform a chemical analysis.
This optical microscope can, by using the double resonance absorption, select a specific molecule and observe absorption and fluorescence caused by a specific optical transition. First, an electron of a valence orbit
2
, owned by the molecule in a ground state illustrated in
FIG. 1
, is excited to a valence orbit
3
that is a vacant orbit by light irradiation, as illustrated in FIG.
2
. This is a first excited state. Next, the electron on a valence orbit
1
is excited, as illustrated in
FIG. 3
, to a hole generated on the valence orbit
2
by irradiating them with a light of another wavelength. This is a second excited state. The molecule then returns to the ground state while emitting fluorescence or phosphorescence, as illustrated in FIG.
4
. And, an absorption image or a luminous image is observed by using the absorption process of
FIG. 2
or the emission of the fluorescence or phosphorescence of FIG.
4
.
At first, when the molecule composing a specimen is to be excited to the first excited state with a light of a resonance wavelength &lgr;
1
by, for example, a laser beam, the number of molecules in the first excited state in a unit volume increases as an intensity of the irradiation light increases. Since a linear absorption coefficient is given as a product of an absorption cross-section per molecule and the number of molecules per unit volume, in the excitation process of
FIG. 3
, the linear absorption coefficient for the light of a resonance wavelength &lgr;
2
subsequently applied depends upon the intensity of the light of the wavelength &lgr;
1
applied first.
In short, the linear absorption coefficient for the wavelength &lgr;
2
can be controlled with the intensity of the light of the wavelength &lgr;
1
. This indicates that, when irradiating a specimen with lights of two wavelengths &lgr;
1
and &lgr;
2
and obtaining a transmission image by the wavelength &lgr;
2
, contrast of the transmission image can be completely controlled with a quantity of the light of the wavelength &lgr;
1
.
On the other hand, when the deexcitation process from the second excited state of
FIG. 3
by fluorescence or phosphorescence is possible, the luminous intensity is proportional to the number of molecules in the first excited state. This makes it possible to control an image contrast, even when used as a fluorescent microscope.
Further, this optical microscope of the prior art is able not only to control the contrast but also to perform the chemical analysis. Since the outermost valence orbit in
FIG. 1
has an energy level intrinsic to a molecule, the wavelength &lgr;
1
is different for the molecule. At the same time, the wavelength &lgr;
2
is also intrinsic to the molecule. As a result, the molecule to absorb or emit a light can be restricted from the two wavelengths &lgr;
1
and &lgr;
2
, so that an accurate chemical composition of a specimen can be identified.
Moreover, when the valence electron is to be excited, only a light having a specific electric-field vector with respect to a molecular axis is intensively absorbed. Thus, if an absorption image or fluorescence image is obtained while determining the directions of polarization of the wavelengths &lgr;
1
and &lgr;
2
, the direction of orientation can also be identified for the same molecule.
In recent years, there has also been proposed (in Japanese Patent Application No. 8-302232) a fluorescent microscope which has a high spatial resolution exceeding a diffraction limit by using the double resonance absorption process.
FIG. 5
is a conceptional diagram illustrating the double resonance absorption process in molecule. It is illustrated in
FIG. 5
that a molecule in the ground state is excited to the first excited state with the light of the wavelength &lgr;
1
and further to the second excited state with the light of the wavelength &lgr;
2
and that fluorescence from this second excited state is extremely weak for some kinds of a molecule.
The molecule having such optical properties experiences a remarkably interesting phenomenon.
FIG. 6
illustrates an extension of a spatial distance in the double resonance absorption process, with an abscissa being an X axis. In
FIG. 6
, there are illustrated a spatial area A
1
which is irradiated with the light of the wavelength &lgr;
2
and a spatial area A
0
which is not irradiated with the light of the wavelength &lgr;
2
. In this spatial area A
0
, a great number of the molecules being in the first excited state are generated by the &lgr;
1
light excitation. At this time, fluorescence emitted with a wavelength &lgr;
3
from the spatial area A
0
can be observed. In the spatial area A
1
, however, the irradiation of the light of the wavelength &lgr;
2
excites most of the molecules in the first excited state instantly to the second excited state at a higher level, so that the molecules in the first excited state disappears. As a result, the fluorescence of the wavelength &lgr;
3
completely disappears, and further, the fluorescence from the second excited state does not exist intrinsically, so that the fluorescence itself is completely inhibited in the spatial area A
1
. It is therefore understood that the fluorescence exists only in the spatial area A
0
.
This result has a remarkably important meaning if considered from the field of application of the microscope. In the scannigng type laser microscope of the prior art, a laser beam is condensed to produce a micro beam thereby to scan a specimen to be observed. At this time, the size of the micro beam is determined by the diffraction limit which in turn is determined by a numerical aperture of a condenser lens and a wavelength, so that a higher spatial resolution cannot be expected on principle. However, according to
FIG. 6
, since the fluorescent area is inhibited with the irradiation of &lgr;
2
, by overlaping the wavelengths of two kinds of &lgr;
1
and &lgr;
2
skillfully, the fluorescent area is made narrower than the size determined by the numerical aperture of the condenser lens and the wavelength, while noticing the irradiation area of &lgr;
1
for example. Thus, the spatial resolution is substantially improved. Therefore, by adopting this principle, it is possible to provide a fluorescent microscope exceeding the diffraction limit. This is a super-resolution microscope using the double resonance absorption process.
In order to enhance the super-resolution of this microscope, another proposal has been made (in Japanese Patent Application No. 9-25444). A molecule of various kinds, which has three quantum states including at least the ground state and in which a thermal relaxation process is more dominant than a relaxation process by fluorescence in transition upon deexcitation from a higher excited state excepting the first excited state to the ground state, is employed as a fluorescence labeler molecule. The specimen in which the fluorescence labeler molecule and a bio-molecule dyed biochemically are chemically bonded, is irradiated with the light of the wavelength &lgr;
1
to excite the fluorescence labeler molecule to the first excited state and
Fujii Masaaki
Iketaki Yoshinori
Omatsu Takashige
Fineman Lee
Japan Science and Technology Corporation
Robinson Mark A.
Wenderoth , Lind & Ponack, L.L.P.
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