Confocal microscope

Details Confocal microscope Confocal microscope

2000-07-06

2002-01-22

Henry, Jon (Department: 2872)

Optical: systems and elements

Compound lens system

With image recorder

C359S369000, C359S389000

Reexamination Certificate

active

06341035

ABSTRACT:

CROSS REFERENCE TO RELATED APPLICATION
This application is based on and claims priority of Japanese Patent Application No. Hei 11-196733 filed on Jul. 9, 1999, the content of which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a microscope. The present invention especially relates to a confocal microscope which properly measures a microscopic structure or a three dimensional structure of a sample.
2. Description of the Related Art
Traditionally, typical confocal microscopes have a disk that has a plurality of pinholes therein. For example, a disk called a ipkow-disk is used as the disk of a disk-scanning confocal microscope. The ipkow-disk has a plurality of pinholes, which are arranged in a spiral on the disk. Furthermore, pinholes of the ipkow disk are spaced a distance of about ten times the pinhole diameter. An example of a confocal microscope using an improved ipkow-disk is described by Juskaitis, T. Wilson et al. fficient real-time confocal microscopy with white light sources Nature Vol. 383, October 1996, pp. 804-806.
FIG. 10
shows a structure of the disk scanning confocal microscope described by T. Wilson et al. This disk-scanning confocal microscope uses a halogen lamp, a mercury lamp etc. as a light source
1
. A collimating lens
2
and a PBS (polarizing beam splitter)
3
are disposed in the optical path of a light beam emitted from the light source
1
. A sample
6
is disposed in a reflected light path of the PBS
3
. The light beam is reflected by the PBS
3
. Then the reflected light beam goes on to the sample
6
through a rotating disk
4
a quarter-wave plate
12
and an objective lens
5
.
The collimating lens
2
, the PBS
3
, the quarter-wave plate
12
and the objective lens form an optical system to direct the light beam emitted from the light source
1
to the sample
6
.
The rotating disk
4
is a random pinhole disk shown in FIG.
11
. This disk has a random pinhole pattern portion
4
a,
an open portion
4
b,
and opaque portions
4
c
and
4
d.
The random pinhole pattern portion
4
a
has a plurality of pinholes, which are arranged on the disk. Furthermore, each pinhole of the disk is at a distance from another pinhole almost equal to the pinhole in diameter. The open portion
4
b
transmits the light beam emitted from the light source
1
. The opaque portions
4
c
and
4
d
are disposed between the random pinhole pattern portion
4
a
and the open portion
4
b.
The rotating disk (rotating object)
4
is made of a transparent circular glass. Low-reflection films made of chrome films are deposited on the transparent circular glass to form opaque portions
4
c
and
4
d.
Likewise, the random pinhole pattern portion
4
a
is made by depositing the low-reflection film (chrome film etc.) on the transparent circular glass except at the location of pinholes. Therefore, each opaque portion
4
c
,
4
d
and the random pinhole pattern portion
4
a
except the pinhole locations will shade the light beam emitted from the light source
1
. The rotating disk
4
is coupled to a rotating shaft
7
, which is coupled to a shaft of a motor so as to rotate shaft
7
at constant speed.
The rotating disk
4
having the pinholes can be replaced with a line pattern disk shown in FIG.
12
. The line pattern disk has a line pattern portion
4
e
instead of the random pinhole pattern portion
4
a
on the transparent circular glass. The line pattern portion
4
e
has a plurality of lines. These lines spaced an almost constant distance each other. These lines are made by depositing the low-reflection film (chrome film etc.) to shade the light beam emitted from the light source
1
. That is, the line pattern portion
4
e
has alternate stripes of light-opaque portions and light-permeable portions (or light-semitransparent portion).
Returning to
FIG. 10
, a focusing lens
8
and a CCD camera
9
are disposed in a transmitted light path of the PBS
3
. The reflected light beam returns toward the PBS
3
, after the light beam emitted from the light source
1
is reflected by the sample
6
. The reflected light beam passes through the PBS
3
, and goes on to the CCD camera
9
through the focusing lens
8
. An image output-terminal of the CCD camera
9
is connected to a computer
10
so as to capture an image. After capturing the image, the computer carried out an image processing so as to display the image on the monitor
11
.
By using the above structure, the light beam emitted from the light source
1
is directed to the PBS
3
through the collimating lens
2
. The light beam reflected by the PBS
3
becomes rays of light incident on the rotating disk
4
which is rotated at constant speed. The light beam passing through the random pinhole pattern portion
4
a
(or the line pattern portion
4
e
) or the open portion
4
b
of the rotating disk
4
becomes a circularly polarized light beam by passing through the quarter-wave plate
12
. Then the circularly polarized light beam is focused on the sample
6
through the objective lens
5
.
The light beam reflected by the sample
6
becomes a polarized light beam which is perpendicular to the light beam incident on the sample
6
by passing through the objective lens
5
and the quarter-wave plate
12
. The polarized light beam passes through the random pinhole pattern portion
4
a
(or the line pattern portion
4
e
) or the open portion
4
b
of the rotating disk
4
again. Then the polarized light beam transits the PBS
3
, and becomes rays of light incident on the CCD camera
9
through the focusing lens
8
.
Now, a timing for taking an image by means of the CCD camera
9
must be synchronized with the rotating speed of the rotating disk
4
so as to capture a composite image and a bright field image. The composite image is captured when the light beam reflected by the sample
6
is passing through the random pinhole pattern portion
4
a
(or the line pattern portion
4
e
). The bright field image is captured when the light beam reflected by the sample
6
is passing through the open portion
4
b.
The composite image comprises a confocal image including non-confocal components. The bright field image comprises a non-confocal image.
That is, an image stored in the CCD
9
is captured in the computer
10
as a composite image during the time of the reflected light beam passing through just about a semi-circular area of the rotating disk
4
including the random pinhole pattern portion
4
a
(or the line pattern portion
4
e
). Next, an image stored in the CCD
9
is captured in the computer
10
as a bright field image during the time the reflected light beam is passing through just about another semi-circular area of the rotating disk
4
including the open portion
4
b.
After that, above two data captured sequentially are processed by a subtractive operation in the computer
10
so as to extract confocal components only. The confocal components are displayed on the monitor
11
as a confocal image. The following describes the subtractive operation with the computer
11
.
(The composite image)−
k
×(the bright field image)=(The confocal image):
k
is constant
Therefore, where the CCD camera
10
is a progressive scanning camera, the composite image is captured while the rotating disk
4
makes a semi-circular rotation thereof. After that, the bright field image is captured while the rotating disk
4
makes a next semi-circular rotation thereof. That is, the composite image and the bright field image are captured sequentially. Then above two captured data are processed by a subtractive operation with the computer
10
so as to extract confocal image data. The confocal image is displayed on the monitor
11
. The above mentioned sequence of processes is made sequentially, and enables to display the confocal image on the monitor
11
.
On the other hand, in case where the CCD camera
10
is an interlaced system camera, an image, which includes a composite image is captured from even-numbered lines of the CCD camera
10
and a bright

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