JP2007101910A - Confocal microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a confocal microscope capable of obtaining a highly accurate multi-color confocal image by correcting displacement of a formed confocal image, resulting from a color aberration caused by returning fluorescence of a different wavelength. <P>SOLUTION: The confocal microscope switches a filter so as to correspond to the multi-wavelength, and thereby obtains the confocal slice image formed by fluorescence returning from a sample excited with the multi-wavelength laser beam. The confocal microscope has a filter means having a plurality of filters differing in thickness in the direction of the optical axis so as to correspond to the multi-wavelengths. The confocal microscope switches a filter of a different thickness according to a corresponding multi-wavelength and thereby obtains each confocal slice image. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a confocal microscope that acquires a confocal slice image of a return fluorescence from a sample excited by a multi-wavelength laser beam by switching a filter corresponding to the multi-wavelength.

  FIG. 3 is a functional block diagram illustrating a system configuration example of a confocal microscope using multi-wavelength laser light. 1 is a sample to be observed, 2 is a stage on which the sample is mounted, and 21 is a stage support. Reference numeral 3 denotes a fluorescence microscope, and 31 denotes an objective lens of the fluorescence microscope arranged to face the sample 1.

  Reference numeral 4 denotes a confocal scanner, which converts multi-wavelength (λ1, λ2,..., Λn) laser light input from a laser light source 5 through a fiber 51 into a beam for scanning focus light on the sample 1. Output to the fluorescence microscope 3. With regard to the confocal scanner, Japanese Patent Application Laid-Open No. 2004-151867 discloses a configuration of a Niipou disk type confocal scanner.

  In order to observe different parts of the sample 1, fluorescent indicators having different wavelengths are used. At that time, the return fluorescence from the sample 1 excited by the multi-wavelength laser light returns to the fluorescence microscope 3 and the confocal scanner 4 and is switched corresponding to the multi-wavelength laser light arranged on the filter wheel 6. Then, the light is imaged in the camera 7 through the filter means of the return fluorescence wavelength, taken into the PC 8 and displayed on the monitor 9.

  The filter means disposed on the filter wheel 6 includes n filters that transmit the wavelengths of the return fluorescence corresponding to the n wavelengths (λ1, λ2,... Λn,) of the laser light, and a control signal M from the PC 8. Thus, the switching operation is performed at a predetermined cycle in time series. This switching cycle is set sufficiently faster than the reaction speed of the sample 1, and the observer can continuously monitor multicolor confocal images without being aware of this switching.

  FIG. 4 is an image diagram showing an optical system of a confocal microscope equipped with a conventional filter means. A confocal image G of the return fluorescence from the sample 1 is imaged in the confocal scanner 4 via the objective lens 31 of the fluorescence microscope 3, and a filter (illustrated) selected by the relay lens 41 and the filter wheel 6. Then, the image is formed in the camera 7 through 62). An image sensor (not shown) is disposed at this imaging position.

  FIG. 5 is a plan view showing an arrangement of n filters provided in the filter wheel 6 in accordance with the return fluorescence wavelengths corresponding to the n wavelengths (λ1, λ2,... Λn,) of the laser light. N filters 61, 62,... 6n having different transmission frequency characteristics are arranged along the circumference of the disk-like filter wheel 6, and are rotationally controlled in a predetermined cycle and are switched to the optical path in time series. The

  The n filters 61, 62,..., 6n transmit the return fluorescence wavelengths corresponding to the n wavelengths (λ1, λ2,. As shown in FIG. 4, the thicknesses of the filters in the optical axis direction are all set to the same thickness T.

Japanese Patent Laid-Open No. 5-60980

  Since the wavelength of the fluorescent indicator covers almost the entire visible region, chromatic aberration always occurs in the objective lens 31 or the relay lens 41, and the imaging position differs for each return fluorescence wavelength. As a result, when multi-color confocal images are superimposed, a color shift occurs, an image different from the actual structure is obtained, and the observation accuracy is lowered.

  FIG. 6 shows an imaging position of the confocal image G in which the return fluorescence having different wavelengths corresponding to the wavelengths λ1 and λ2 of the laser beam forms an image in the camera 7 through the filters 61 and 62 having the same thickness T. It is an image figure which shows deviation | shift.

  The present invention has been made to solve the above-described problems, and corrects an imaging position shift of a confocal image caused by chromatic aberration due to return fluorescence of different wavelengths, thereby providing a highly accurate multicolor confocal image. The objective is to realize a confocal microscope capable of acquiring

In order to achieve such a subject, the present invention has the following configuration.
(1) In a confocal microscope that acquires a confocal slice image of a return fluorescence from a sample excited by a multi-wavelength laser beam by switching a filter corresponding to the multi-wavelength,
A filter unit having a plurality of filters having different thicknesses in the optical axis direction corresponding to the multi-wavelengths, and acquiring confocal slice images by switching the filters having different thicknesses corresponding to the multi-wavelengths. A confocal microscope characterized by this.

(2) When the thickness of the filter is T and the refractive index of the material is N, the moving amount P of the image formation position of the confocal slice image is given by P = (1-1 / N) T (2) The confocal microscope according to (1).

(3) The confocal microscope according to (1) or (2), wherein the imaging positions of the confocal slice images corresponding to the multiple wavelengths are maintained at the same position.

(4) The filter means is characterized in that a plurality of filters having different thicknesses are arranged along the circumference of a disk-like filter wheel, and the filters are switched in time series by being controlled in rotation at a predetermined period. The confocal microscope according to any one of (1) to (3).

As is apparent from the above description, the present invention has the following effects.
(1) Chromatic aberration due to different wavelengths of return fluorescence can be corrected by changing the thickness of the filter corresponding to this wavelength, and all confocal images can be formed at the same position.

(2) As a result, the occurrence of color misregistration when multi-color confocal images are overlaid is eliminated, so that it is possible to observe a multi-color confocal image with high accuracy using multi-wavelength laser light.

  Hereinafter, the present invention will be described in detail with reference to the drawings. FIG. 1 is an image diagram showing an optical system of a confocal microscope equipped with a filter means to which the present invention is applied. The same elements as those described in FIGS. 3 to 6 are denoted by the same reference numerals, and description thereof is omitted. Hereinafter, the characteristic part of the present invention will be described.

  In FIG. 1, filters 601 (for λ1), 602 (for λ2),... 60n (for λn) to which the present invention is applied are arranged along the circumference of the filter wheel 600 and controlled as in FIG. The rotation is performed by the signal M.

  The feature of the present invention is that the filter 601 (for λ1), 602 (for λ2),... 60n (for λn) has a thickness in the optical axis direction such as T1, T2,. It is in the point provided with different thickness corresponding to.

  The present invention utilizes the optical characteristic that the image formation position of the confocal image G moves depending on the thickness T in the optical axis direction of the filter, and shifts the image formation position corresponding to each return fluorescence wavelength due to chromatic aberration. As a result, the imaging position corresponding to each return fluorescence wavelength is maintained at a fixed position.

In general, when a filter having a thickness T is inserted between the relay lens 41 and the camera 7, the imaging position moves by a distance P calculated by the following equation, compared to a case where no filter is inserted.
Moving amount of image forming position P = (1-1 / N) T (1)

  Here, N represents the refractive index of the filter material. Since the refractive index n varies depending on the wavelength, if the filter thickness T is constant, the moving amount P of the imaging position varies with the wavelength, and the imaging position shifts due to the chromatic aberration described above.

  In the present invention, the filter thickness T corresponding to the refractive index N for each wavelength is used so that the amount of movement of the imaging position corresponding to the wavelength of each return fluorescence becomes equal using the characteristic of the equation (1). Is changed to correct the image formation position and maintain it at a fixed position.

  FIG. 2 is an image diagram for explaining that confocal images G of return fluorescence having different wavelengths corresponding to different wavelengths of laser light are imaged at the same position in the camera 7 through filters having different thicknesses.

  FIG. 2A shows an imaging position of the confocal image G by the filter 601 having the thickness T1 for the return fluorescence wavelength corresponding to the wavelength λ1 of the laser light. FIG. 2B shows the imaging position of the confocal image G by the filter 602 having a thickness T2 for the wavelength of the return fluorescence corresponding to the wavelength λ2 of the laser beam. These image positions match.

  In FIG. 2A, if the thickness T1 of the filter 601 is a conventional configuration equal to the thickness T2 of the filter 602, the return fluorescence confocal image G ′ by the wavelength λ1 of the laser light is indicated by a dotted line. Thus, the position of G is shifted. According to the present invention, this shift is corrected, and the imaging positions of the confocal images G of the return fluorescence with all laser light wavelengths are maintained at the same position.

It is an image figure which shows the optical system of the confocal microscope provided with the filter means to which this invention is applied. It is an image figure explaining that the confocal image of the return fluorescence of a different wavelength corresponding to a different wavelength of a laser beam forms in the same position in a camera through a filter with different thickness. It is a functional block diagram which shows an example of the system configuration | structure of a confocal microscope using a multiwavelength laser beam. It is an image figure which shows the optical system of the confocal microscope provided with the conventional filter means. It is a top view which shows the arrangement | sequence of the filter with which a filter wheel is equipped corresponding to the return fluorescence by the laser beam of multiple wavelengths. It is an image figure which shows the imaging position shift of the confocal image which the return fluorescence of a different wavelength corresponding to a different wavelength of a laser beam forms in a camera through the filter which has the same thickness.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Sample 3 Fluorescence microscope 31 Objective lens 4 Confocal scanner 41 Relay lens 7 Camera 600 Filter wheel 601,602, ... 60n Filter T1, T2, ... Tn Filter thickness G Confocal image

Claims (4)

In the confocal microscope that acquires the confocal slice image of the return fluorescence from the sample excited by the laser light of multiple wavelengths by switching the filter corresponding to the multiple wavelengths,
A filter unit having a plurality of filters having different thicknesses in the optical axis direction corresponding to the multi-wavelengths, and acquiring confocal slice images by switching the filters having different thicknesses corresponding to the multi-wavelengths. A confocal microscope characterized by this.   When the thickness of the filter is T and the refractive index of the material is N, the moving amount P of the imaging position of the confocal slice image is given by P = (1-1 / N) T. The confocal microscope according to claim 1.   3. The confocal microscope according to claim 1, wherein an imaging position of the confocal slice image corresponding to the multiple wavelengths is maintained at the same position. The filter means is characterized in that a plurality of filters having different thicknesses are arranged along the circumference of a disk-shaped filter wheel, and the filters are switched in time series by being controlled to rotate at a predetermined period. The confocal microscope according to any one of 1 to 3.

Images