Optics: measuring and testing – By light interference – For dimensional measurement
Reexamination Certificate
2000-04-03
2002-12-17
Lee, John R. (Department: 2881)
Optics: measuring and testing
By light interference
For dimensional measurement
C356S072000, C356S073100, C356S317000, C356S318000, C356S417000, C356S484000, C356S485000, C356S490000, C356S497000, C250S227180
Reexamination Certificate
active
06496267
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to scanning microscopes. More particularly, the present invention relates to a scanning microscope which allows morphological observation and fluorescence observation to be performed simultaneously and enables both a morphological observation image and a fluorescence observation image to be obtained within a reduced period of time.
A technique called “low-coherence interferometry” such as that disclosed in U.S. Pat. No. 5,321,501 is known as a method that allows observation of the inside of an opaque scattering sample, e.g. a biological tissue.
FIG. 11
shows a typical optical system for the low-coherence interferometry. Light from a light source
81
with a short coherence length is split by a beam splitter
82
between a signal light path leading to a sample
5
and a reference light path leading to a reflecting mirror
83
. Light going and returning along the signal light path and the reference light path are recombined in the beam splitter
82
. At this time, because the signal light path forms an optical path length substantially equal to that of the reference light path at an observation position
6
within the sample
5
, only light scattered back from a region at the observation position
6
within a range in the optical axis direction that is substantially equal to the coherence length interferes with the reference light. Accordingly, by detecting the resulting interference signal with a detector
84
, information about the inside of the sample
5
can be selectively obtained in the optical axis direction. In general, the reflecting mirror
83
in the reference light path is moved in the optical axis direction, thereby performing scanning in the direction of depth of the sample and, at the same time, giving a Doppler shift to the reference light. The low-coherence interferometry generally includes heterodyne interferometric measurement that is carried out to detect a beat signal having a Doppler shift frequency in the interference signal. Therefore, the measurement can be performed with a very high S/N ratio. Accordingly, if near infrared light or the like is used as the light source
81
, it is possible to detect feeble scattered light from a position as deep as several millimeters from the surface of the scattering sample
5
. By scanning the signal light or the sample
5
in a plane perpendicular to the optical axis, it is possible to obtain an image of a section perpendicular to the optical axis.
Meanwhile, a low-coherence interferometric method is published in “Journal of Modern Optics”, Vol. 45, No. 4, p.765 (1998), in which acoustooptic devices are disposed in the signal light path and the reference light path, respectively, and a beat signal corresponding to the difference between the modulation frequencies of the acoustooptic devices is detected without moving the reflecting mirror.
Incidentally, a fluorescence observation method is known as an observation method for biological samples or the like. According to the fluorescence observation method, a cellular tissue or a specific substance is labeled with a fluorescent dye, and a fluorescence image produced when excitation light is applied to the sample is observed. The sample may be sliced for microscopic observation. Recently, however, there have been increasing needs to observe biological samples or the like in a living state, and there has been a growing demand for obtaining a fluorescence image at some depths from the sample surface.
During fluorescence observation, it is desirable to be possible to simultaneously obtain morphological information about a fluorescence-labeled tissue or substance and information concerning a surrounding spatial structure. However, it is difficult to obtain information about the inside of a thick sample in ordinary microscopic observation. In observation of such morphological information, for example, when it is intended to observe changes of biological activities of a living biological tissue with time, it is desirable that the time required for the observation should be as short as possible. When morphological observation or the like is performed simultaneously with fluorescence observation, if excitation light is continuously applied to the fluorescence-labeled sample for a long period of time, the fluorescent dye fades. Consequently, the fluorescence image becomes dark as time goes by. Therefore, in this case also, the time required for observation should be minimized.
The above-described patent and literature give no description of a fluorescence observation method and do not mention an arrangement in which low-coherence interferometric measurement is carried out during fluorescence observation. Such an arrangement is disclosed in U.S. patent application Ser. No. 09/172,676 and Japanese Patent Application Unexamined Publication (KOKAI) No. 11-119106, which were filed by the present applicant. However, the disclosed arrangement uses a method in which the reflecting mirror in the reference light path is moved to perform observation. Therefore, it is necessary to move the reflecting mirror also when observing a section within the sample that is perpendicular to the optical axis. Accordingly, when low-coherence interferometric measurement is carried out simultaneously with fluorescence microscopic observation, in which, generally, a section perpendicular to the optical axis is observed, the time required for measurement undesirably lengthens by an amount corresponding to the time needed for mechanical drive of the reflecting mirror.
SUMMARY OF THE INVENTION
In view of the above-described problems with the prior art, an object of the present invention is to provide a scanning microscope in which when performing fluorescence observation of the inside of a thick sample or the inside of an opaque scattering sample, it is possible to simultaneously perform morphological observation for obtaining morphological information or the like in the same region of interest as that for the fluorescence observation, and it is possible to obtain both a fluorescence observation image and a morphological observation image within a reduced period of time.
To attain the above-described object, the present invention provides a scanning microscope including a low-coherence light source and a device for splitting low-coherence light from the low-coherence light source between a first optical path and a second optical path. A frequency modulator is placed in at least one of the first and second optical paths to produce a frequency difference between light passing through the first optical path and light passing through the second optical path without changing the optical path length of each optical path. An objective optical system is placed in the first optical path to apply light to a sample and to collect light from the sample. A scanning device is placed in the first optical path to scan the sample and the light applied by the objective optical system relative to each other in a plane perpendicular to the optical axis of the objective optical system. The scanning microscope further includes a device for combining together the first and second optical paths, and an interference signal detecting system for detecting an interference signal having the frequency difference from the combined light. A fluorescence branching device branches fluorescence from the sample excited by the low-coherence light. A fluorescence detecting system detects the fluorescence branched by the fluorescence branching device.
The arrangement and operation of the scanning microscope according to the present invention will be described below with reference to
FIG. 1
, which shows the arrangement of the scanning microscope according to the present invention.
Low-coherence light from a low-coherence light source
1
is split between a first optical path and a second optical path by an optical path splitting device
2
. In
FIG. 1
, an optical path along which light reflected by the optical path splitting device
2
travels is defined as a first optical path, and an o
Hashmi Zia R.
Lee John R.
Olympus Optical Co,. Ltd.
Pillsbury & Winthrop LLP
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