Optics: measuring and testing – By dispersed light spectroscopy – With sample excitation
Reexamination Certificate
2000-10-20
2002-10-01
Stafira, Michael P. (Department: 2877)
Optics: measuring and testing
By dispersed light spectroscopy
With sample excitation
Reexamination Certificate
active
06459484
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a scanning optical apparatus which is capable of detecting a plurality of beams of emission light (fluorescent light) simultaneously.
2. Description of Related Art
In general, fluorescence photodetectors are used in many fields including medicine and biology for the purpose of detecting protein and genes in which living tissues and cells are labeled by fluorescence. In particular, a multiple-fluorescence detection technique that a specimen stained by a plurality of fluorescent dyes is observed at a time has recently been used to exercise its power for the analysis of a gene and the clarification of an intracellular structure.
As an effective means of such fluorescence detection, a laser scanning microscope (LSM) is well known.
FIG. 1
shows a typical arrangement of an optical system of the LSM for fluorescence. In this LSM, laser beams emitted from three laser oscillators is
101
a
,
101
b
, and
101
c
which have different oscillation wavelengths are combined on a common optical path by laser-beam combination dichroic mirrors
102
a
and
102
b
. After that, a combined laser beam is enlarged to a beam diameter of proper size through a beam expander
103
and is reflected by a dichroic mirror
104
. The laser beam is then deflected by an X-Y scanning optical system
105
, and after being collected through a pupil relay lens
106
and an objective lens
107
, irradiates a specimen
108
. The specimen
108
is thus scanned with a laser spot, and emission light from the specimen
108
, excited by the irradiation of the laser beam, follows a path from the objective lens
107
to the dichroic mirror
104
, and after being transmitted through the dichroic mirror
104
, is separated by a dichroic mirror
109
a
for separation. Emission light reflected by the dichroic mirror
109
a
is condensed by an imaging lens
110
a
and passes through a confocal aperture
111
a
. After light with wavelengths other than that of the first emission light required is absorbed or reflected by an emission filter
112
a
, the intensity of the first emission light is detected by a photodetector
113
a
. The confocal aperture
111
a
is placed at a position optically conjugate with the focal point of the objective lens
107
and blocks light other than the first emission light excited by the laser spot. An image thus obtained has a very high contrast. Moreover, a distance between the specimen
108
and the objective lens
107
is relatively changed along the optical axis, and thereby a three-dimensional image can be obtained.
On the other hand, emission light transmitted through the dichroic mirror
109
a
is further separated by a dichroic mirror
109
b
. Emission light reflected by the dichroic mirror
109
b is condensed by an imaging lens
110
b
and passes through a confocal aperture
111
b
. Through an emission filter
112
b
transmitting only the second emission light required, the intensity of the second emission light is detected by a photodetector
113
b
. Emission light transmitted through the dichroic mirror
109
b
is condensed by an imaging lens
110
c
and passes through a confocal aperture
111
c
. Through an emission filter
112
c
transmitting only the third emission light required, the intensity of the third emission light is detected by a photodetector
113
c
. The optical system mentioned above is capable of detecting simultaneously triple-excitation emission light with three wavelengths emitted from the laser oscillators
101
a
,
101
b
, and
101
c
. Whenever the conditions of multiple excitation, such as wavelengths of laser beams, the kind of fluorescent dye, and the number of excitation laser oscillators, are changed, the dichroic mirror
104
, the dichroic mirrors
109
a
and
109
b
, and the emission filters
112
a
,
112
b
, and
112
c
are replaced with filters having the optimum dispersion characteristics.
However, a conventional LSM for fluorescence using these optical filters has the following problems. First, the optical filters, because of their fabrication restrictions, cannot be designed to determine dispersion characteristics at will, and thus the amount of emission light and the S/N ratio are limited. In particular, the emission filter must completely block excitation light, but a filter that sufficiently transmits light in the wavelength region of the highest fluorescence intensity, close to the wavelength of the excitation light, cannot be fabricated at present. Second, expensive optical filters which are exclusively used in accordance with the wavelength of the excitation light and the fluorescent dye must be prepared. When a variety of multiple excitation are taken into account, it is unavoidable to cause an increase in the number of filters and the complication and oversizing of an apparatus used. Third, in the optical system of the LSM for fluorescence, multiple fluorescence is dispersed through a plurality of optical filters, and hence an appreciable amount of light is lost before emission light reaches each of the photodetectors. Any of these problems becomes severe as the multiplicity of excitation light and emission light increases.
In order to solve the above problems, techniques of selecting and detecting a plurality of fluorescence wavelengths without using the optical filters are proposed. For example, WO 95/07447 discloses a spectroscope and a confocal fluorescence microscope in which a light beam decomposed into a wavelength spectrum by a prism is dispersed into a first wavelength region transmitted through a slit-like mirror and a second wavelength region reflected thereby, and the position and width of a second slit restricting the slit-like mirror and the second wavelength region are controlled so that two arbitrary wavelength regions can be selected and detected. On the other hand, Japanese Patent JP-A-2000-199855 discloses a scanning optical apparatus in which emission light transmitted through a confocal aperture is decomposed by a prism into a wavelength spectrum, which is received by a light-deflecting microelement array such as a digital mirror array (DMD). In this case, each of light-deflecting microelements has light-deflection angles that cause emission light to be received by a plurality of photo-detectors, and the light-deflection angles are arbitrarily selected so that the optimum fluorescence detection is always made with respect to various combinations of wavelengths of excitation light and fluorescent dyes and a multiple fluorescence image with a high S/N ratio can be obtained. In these techniques, however, filters for the separation of emission light are merely dispensed with, and a dichroic mirror for excitation cannot be removed.
U.S. Pat. No. 5,751,417, by contrast, discloses a confocal LSM which dispenses with the dichroic mirror for excitation. In this LSM, incident excitation light is transmitted through a slit array and is separated in wavelength by a first spectroscope. Separated excitation light is projected at the position where the light is reflected by a wavelength selective member which is a slit array transmitted through emission light and reflecting excitation light. Reflected excitation light is projected on a confocal slit array by a second spectroscope which is identical with the first spectroscope, and irradiates a specimen through an imaging lens, a scan mirror, and an objective lens. Emission light produced by the irradiation of the excitation light follows a reverse course, and after being transmitted through the confocal slit array and separated in wavelength by the second spectroscope, is projected on the wavelength selective member. Here, since the wavelength of emission light is transmitted and the wavelength of excitation light is reflected, the excitation light reflected by the specimen and the emission light are separated. The emission light transmitted through the wavelength selective member is spatially returned to an original wavelength spread by a third spectroscope which is identical with the first and second spectros
Olympus Optical Co,. Ltd.
Pillsbury & Winthrop LLP
Stafira Michael P.
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