Optical: systems and elements – Compound lens system – Microscope
Reexamination Certificate
2001-08-21
2003-10-14
Sikder, Mohammad (Department: 2872)
Optical: systems and elements
Compound lens system
Microscope
C359S387000, C359S388000, C359S389000
Reexamination Certificate
active
06633432
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to an optical device and microscope. In particular, the present invention relates to a microscope-spectrometer device, such as a super-resolution fluorescence microscope using light of two different wavelengths, that improves detection sensitivity and spatial resolution by concentrating light at a sample surface using the wavefronts of light of two wavelengths.
2. Description of the Related Art
Technology of optical microscopy has long been developed, resulting in the invention of various types of microscopes. Moreover, microscope systems with improved performance have been developed in recent years due to advances in related technologies beginning with laser technology and electron imaging technology.
Within this context, for example, Publication of Unexamined Patent Application No. Hei 8-184552 has proposed a high performance microscope capable of chemical analysis, in addition to control of contrast of the obtained image, by the use of a double-resonance absorption process resulting from illumination of the sample by light of multiple wavelengths.
The principles of this microscope, which operates by selection of a particular molecule by use of a double-resonance absorption process to observe absorption and fluorescence due to particular optical transitions, will be explained while referring to FIG.
9
-FIG.
17
.
FIG. 9
shows the electron structure of a valence electron orbital of a molecule comprising the sample. First an electron of a valence electron orbital of a molecule in a ground state (S
0
) as indicated in
FIG. 9
is excited by light into an excited state (S
1
state) as shown in FIG.
10
. Then light of another wavelength causes excitation in the same manner, resulting in a second excited state (S
2
state) shown in FIG.
11
. The molecule returns to the ground state indicated by
FIG. 12
after light emission by fluorescence or phosphorescence.
Microscopy utilizing double-resonance absorption observes an absorption image and an emitted light image using the absorption processes of FIG.
10
and the fluorescence or phosphorescence process of FIG.
11
. Although this microscopy method first excites a molecule comprising the sample into the S
1
state as shown in
FIG. 10
by light of a resonant wavelength &lgr;
1
due to laser light, etc., the number of molecules in the S
1
state per unit volume increases with the intensity of the irradiating light.
Here the line absorption coefficient is given by the product of the absorption cross section per molecule and the number of molecules per unit volume. Then the line absorption coefficient with respect to illuminating resonant wavelength &lgr;
2
during the process of
FIG. 11
depends upon the intensity of light of the initial irradiating wavelength &lgr;
1
. That is to say, the absorption coefficient with respect to &lgr;
2
becomes controllable by the intensity of light of &lgr;
1
. This indicates that transmission image contrast can be entirely controlled by light of wavelength &lgr;
1
if the sample is irradiated with light of the two wavelengths, wavelength &lgr;
1
and wavelength &lgr;
2
, and if the transmission image due to wavelength &lgr;
2
is imaged.
Moreover, if de-excitation is possible from the excited state of
FIG. 1
by fluorescence or phosphorescence, the intensity of such light emission is proportional to the number of molecules in the S
1
state. Therefore it becomes possible to control image contrast even during use as a fluorescence microscope.
Furthermore, microscopy using double-resonance absorption is capable of chemical analysis and isn't simply limited to the above mentioned control of image contrast. That is to say, the outermost valence electron orbital shown in
FIG. 9
has inherent energy levels for each molecule. Therefore wavelength &lgr;
1
depends upon the molecule, and simultaneously, &lgr;
2
also is characteristic of the molecule.
Here, although it is possible to observe an absorption or fluorescence image to a certain extent even when irradiation occurs at the conventional single wavelength, such observation is generally impossible until precise analysis is carried out of the chemical composition of the sample since wavelength regions coincide for any number of molecular absorption bands.
In contrast, since a microscopy using double-resonance absorption is limited to molecules emitting light or absorbing light at the two wavelengths &lgr;
1
and &lgr;
2
, it becomes possible to determine chemical composition of the sample with greater accuracy than by the conventional method. Moreover, since absorption is intense when a valence electron is excited only for light that has an electrical field vector along the polarization direction of wavelength &lgr;
1
and wavelength &lgr;
2
, it becomes possible to even analyze the orientation direction of the same molecule if polarization direction of wavelength &lgr;
1
and wavelength &lgr;
2
are determined and then an absorption or a fluorescence image is taken.
Moreover, recently (e.g., Publication of Unexamined Patent Application No. Hei 10-142151) a fluorescence microscope has been proposed that has high spatial resolution that exceeds the diffraction limit by use of double-resonance absorption.
FIG. 13
is a conceptual drawing showing the process of double-resonance absorption that occurs in a molecule. The molecule in the ground state S
0
is excited to S
1
, which is a first excited state, due to light at wavelength &lgr;
1
. Furthermore, this shows excitation to S
2
, which is a second excited state, due to light at wavelength &lgr;
2
. This also shows the case of extremely weak fluorescence from S
2
.
Extremely interesting phenomena occur in the case of a molecule that has the optical properties indicated in FIG.
13
.
FIG. 14
is a conceptual drawing of the double-resonance absorption process, in the same manner as
FIG. 13
, wherein the horizontal X axis indicates width of spatial distance, spatial region A
1
is irradiated by light of wavelength &lgr;
2
, and spatial region A
0
isn't irradiated by light of wavelength &lgr;
2
.
Within
FIG. 14
, numerous molecules are generated in the S
1
state due to excitation by light of wavelength &lgr;
1
at spatial region A
0
, and then fluorescence is visible due to light emission at wavelength &lgr;
3
from spatial region A
0
. However, since spatial region A
1
is irradiated by light of wavelength &lgr;
2
, most molecules in the S
1
state are immediately excited to the high S
2
state such that molecules in the S
1
state aren't present. This type of phenomenon is confirmed for any number of molecules. By this means, even if fluorescence of wavelength &lgr;
3
entirely disappears, fluorescence itself at the A
1
region becomes entirely controllable since there was no fluorescence originally from the S
2
state. Therefore fluorescence occurs only in the A
0
spatial region.
This result has extremely important meaning when considered from the standpoint of the applied field of microscopy. That is to say, a conventional scanning-type laser microscope, etc. concentrates light into a micro-beam by means of a condensing lens and then scans across the observed sample. During this process, micro-beam size becomes that of the diffraction limit determined by wavelength and the numerical aperture of the condensing lens, and spatial resolution better than this limit can't be anticipated.
However, in the case of
FIG. 14
, lights of two types (wavelength &lgr;
1
and wavelength &lgr;
2
) are skillfully combined spatially, and the fluorescence region is controlled by irradiation of light of wavelength &lgr;
2
. Therefore, for example, upon consideration of the region of irradiation of light of wavelength &lgr;
1
, the fluorescence region can be made more narrow than even that of the diffraction limit determined by wavelength and numerical aperture of the condensing lens. Therefore this principle can be utilized to make possible a super-resolution microscope (e.g. a fluorescence microscope) us
Frishauf Holtz Goodman & Chick P.C.
Olympus Optical Co,. Ltd.
Sikder Mohammad
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