The present disclosure pertains, inter alia, to slit-scanning confocal microscopes.
In a conventional slit-scanning confocal microscope, light emitted from a light source passes through a first slit. An image of the first slit is used as scanning light that passes through a scanning optical system that forms an image of the first slit on a sample using an objective lens. Reflected light or fluorescence light from the sample passes back through the objective lens and scanning optical system to convert the reflected light or fluorescence light into non-scanning light. The reflected light or fluorescencet light passes through a second slit disposed optically conjugate with the first slit. The intensity of the light passing through the second slit is measured by a detector (line sensor) to obtain image data. A galvanic mirror or the like is used in the scanning optical system to perform one-dimensional scanning of light in a direction normal to the length direction of the slit.
It is not always necessary to utilize a scanning optical system. Alternatively, scanning may be performed by moving an integrally arranged light source, illumination optical system, imaging optical system, slits, or the like.
Detailed description of a laser confocal scanning microscope is given in Tsuruta, “Light Pencil,” chapter 5 of New Technology Communications, pp. 177-205. In the slit-scanning confocal microscope, a pinhole used in a laser confocal scanning microscope is replaced by a slit.
In a conventional confocal microscope, the sample is scanned with a beam of light from a pinhole-like light source. Only light that has passed through a pinhole, situated conjugate to the light source, is detected. Hence, this conventional confocal microscope has high resolving power in the depth dimension of the sample. However, images and image data are produced very slowly because the scanning is only two-dimensional using the very narrow beam from the pinhole.
To increase the speed of operation of a conventional laser confocal microscope using a scanned beam from a point light source, a slit-scanning confocal microscope can be used and are available. However, conventional slit-scanning confocal microscopes exhibit reduced resolution (lower resolving power) in the depth dimension of the sample.
The present invention addresses the shortcomings of conventional devices summarized above. The invention provides, inter alia, slit-scanning confocal microscopes having enhanced resolving power in the sample's depth dimension.
A first embodiment for solving the problems summarized above is a slit-scanning confocal microscope that includes a slit-like light source, an illumination optical system, and an imaging optical system. The illumination optical system forms an image of the light source on a sample, and the imaging optical system forms an image of reflected light, transmitted light, or fluorescence light from the sample on a line sensor. The line sensor is disposed in a position that is optically conjugate with the light source. The light source is divided into multiple unit light sources. Each unit light source has a size and location that are optically conjugate with a respective pixel of the line sensor.
A second embodiment is similar to the first embodiment, but the light source is controlled such that the unit light sources are individually lit in a predetermined, ordered manner.
A third embodiment is similar to the second embodiment, but the light source is controlled such that the unit light sources adjacent to a lit unit light source are turned off, and unit light sources adjacent to an non-lit unit light source are lit.
A fourth embodiment is similar to the second or third embodiments but the light source is controlled such that the unit light sources that are lit and the unit light sources that are not lit are arranged alternatingly relative to each other.
A fifth embodiment is similar to any of the second, third, or fourth embodiments but includes a signal-operation unit. Assuming that Sa is an output signal obtained from a pixel in the line sensor when the unit light source that is conjugate with the pixel is lit, and assuming that Sb is an output signal obtained from the pixel in the line sensor when the unit light source that is conjugate with the pixel has been turned off, while the unit light sources on both sides of the turned-off unit light source are lit. A difference output signal (Sa−Sb) is used as a corrected output signal of the output signal Sa of the pixel in the line sensor.
a) is a schematic diagram illustrating the principle of a slit-scanning confocal microscope according to an embodiment of the invention;
b) is a view illustrating an array of unit light sources and a line sensor;
An embodiment of the present invention will be described below with reference to the drawings.
A light-source plane is defined by the slit-like light source 1 (which may be used as a secondary light source), and the plane is conjugate with the image plane on a sample 3. An illumination optical system 2 makes an image of the slit-like light source 1 on the image plane (xs-ys plane) on the sample 3 using light emitted from the slit-like light source 1. This light that is incident on the sample 3 can produce any of various types of light, including fluorescence light, reflected light, or transmitted light (transmitted light is illustrated in
The position of the line sensor 5 is conjugate to the slit-like light source 1. The detection plane of the line sensor 5 is conjugate to the image plane. Therefore, light from the image plane (xs-ys plane) on the sample 3 is focused on the line sensor 5 (xd-yd plane). The sample 3 is scanned in the optical-axis direction and in the xs-direction to provide image data over the entire target sample region. Alternatively, the slit-like light source 1 may be moved in the x-axis direction and the line sensor 5 may be moved in the xd-axis direction while the conjugate relationship is maintained between the slit-like light source 1 and the line sensor 5.
b) illustrates the positional relationship between the slit-like light source 1 and the pixels of the line sensor 5. The slit-like light source 1 is divided into multiple unit light sources. Each unit light source is sized and positioned so that it is optically conjugate to a respective pixel of the line sensor 5 via the illumination optical system 2 and the imaging optical system 4.
a)-2(b) illustrate an exemplary relationship between the unit light sources, sample regions, and the pixels of the line sensor. The manner of indicating the slit-like light source and the pixels is similar to that used in
In
Light propagation will be described with reference, for example, to the unit light source SA and the pixel D0. In the lighting condition shown in
Similarly, in the lighting condition shown in
In the following description, it is assumed that Sa is an output signal supplied from the pixel D0 in the lighting condition shown in
The portion on the Xs-axis in the region A of
Then, the unit light sources SB1 and SB2 adjacent to the unit light source SA are lit, as illustrated in
In
Similarly, in
As described above, in the lighting situation shown in
Therefore, whenever a difference output signal (Sa−Sb) is obtained during operation, the difference output signal (Sa−Sb) includes information of a region G of
As illustrated in
In the above description, the unit light sources of the slit-like light source 1 are arranged such that lit ones and turned-off ones are arranged alternatingly. However, this alternating arrangement is not always necessary. For example, the unit light sources can be discretely lit in any order, and the same advantageous effects are obtained when the difference output signal (Sa−Sb) is used as the corrected output signal of the output signal Sa of a pixel in a line sensor. Here, it is assumed that Sa is an output signal of a pixel in the line sensor that is conjugate with the lit unit light source, and Sb is an output signal of the same pixel when unit light sources adjacent to the unit light source are lit.
In
Number | Date | Country | Kind |
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JP 2007-033397 | Feb 2007 | JP | national |
This application is a continuation of, and claims priority to and the benefit of, PCT/JP/2008/052123, filed on Feb. 8, 2008, and published as WO 2008/099778 on Aug. 21, 2008, which claims priority to and the benefit of Japan Patent Application No. 2007-033397, filed on Feb. 14, 2007, all of which being incorporated herein by reference in their respective entireties.
Number | Date | Country | |
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Parent | PCT/JP2008/052123 | Feb 2008 | US |
Child | 12541858 | US |