WO2006109561A1

WO2006109561A1 - Microscope imaging apparatus and method

Info

Publication number: WO2006109561A1
Application number: PCT/JP2006/306256
Authority: WO
Inventors: Hiroyuki Ogino
Original assignee: Kyoto University
Priority date: 2005-04-07
Filing date: 2006-03-28
Publication date: 2006-10-19
Also published as: JPWO2006109561A1; JP5062588B2

Abstract

There are provided a microscope imaging apparatus and a method for acquiring a bright image with high resolution and high depth. A unit multi-luminous point light source (5) is provided at a position for forming an image with uniform illumination. The light source (5) illuminates the entire surface of a sample (9) uniformly and illuminates the sample (9) a plurality of times for each illumination group with coherent light or partial coherent light. A calculation section (10) compounds an image acquired at an imaging section (8) in correspondence with the sample (9) illuminated by the illumination group, thus composing a sheet of image. The illumination group has one or a plurality of bright parts not continuous to each other and dark parts surrounding the bright parts. The entire surface of the sample is illuminated by all the bright parts of the illumination group.

Description

Specification

Microscope imaging apparatus and method

Technical field

[0001] The present invention relates to a microscope imaging apparatus and method for observing and imaging with high resolution and depth.

Background art

[0002] Conventional microscopes are mainly intended for observation, and in order to observe brightly and at a high magnification, contrivances have been made to make the illumination uniform and increase the numerical aperture (NA). The elements that make up a microscope can be broadly divided into (1) an illumination system and (2) an objective observation system. A microscope is an apparatus that enables these specimens to be observed and imaged under optimum conditions.

[0003] Illumination plays a role as a probe to obtain sample force optical information, and since a microscope can produce an optimal light source for an artificial purpose, it is incoherent, partially coherent, or coherent. Band power Able to irradiate light of any condition up to light of a single wavelength.

[0004] The optical resolution, which is the main performance of the objective observation system, depends on the nature and wavelength of light, and the theoretical resolution limit is 2NAZ for incoherent light and NA Ζ λ for coherent light. The parameter that can increase the resolution here is to increase the aperture when the wavelength of light is determined.Specifically, use an optical material that has a large magnification and refractive index of the objective lens and good wavelength characteristics. Was that.

Conventionally, the observation of an image formed by an optical system has been mainly the observation with the naked eye. For this reason, the performance of the illumination system is focused on the illumination suitable for the detection of the image by the eye. The role played by is as follows.

(1) Illuminate the sample uniformly.

(2) Illuminate the aperture of the objective lens uniformly.

(3) Make sure that light does not enter the objective lens without contributing to image formation.

Illumination methods that make this possible include:

(a) Relay capacitor system (b) Kerala lighting

(c) Critical lighting

Among them, the best illumination method is Koehler illumination (for example, Non-Patent Document 1).

[0006] Kohler illumination is telecentric on the image side and theoretically has a Lambertian surface with uniform brightness at infinity, which is an aperture stop on the front focal plane of the lens and a sample (covered) on the rear focal plane. Place the lens so that it is the illumination surface) and illuminate it.

[0007] However, the actual light source does not have a theoretically uniform Lambertian surface. For example, the luminance of a light emitting part of a tungsten filament, a mercury discharge lamp, etc. is uneven with respect to location and direction. This causes illumination spots.

In recent years, photographs and image sensors have been used instead of eyes when detecting images. As a result, more uniform illumination was required. Therefore, in order to illuminate uniformly, a fly's lens called an integrator is further arranged between the collector lens and condenser lens of the Koehler illumination, and the specimen surface is uniformly illuminated by each unit lens. , Their combined total illumination becomes uniform. (For example, Patent Document 1)

[0009] In addition, the resolution limit of both the illumination system and the objective optical system is λ ΝΑ2ΝΑ, that is, 2ΝΑΖ λ (lines / mm) for incoherent light, and λ ΖΝΑ, that is, ΝΑ / λ (lines) for coherent light. / mm). Partially coherent light is in-between but non-linear, and diffracted light extends to λ Z4NA. The basis of the resolution is based on the ability to identify the naked eye as a detector, and it is still the most powerful detector of humanity, and it is an image information input and processing device. Absent.

[0010] Generally, in a microscope, a transmission function is determined by integrally acting an illumination system and an objective optical system, and illumination can be changed to incoherent light power or partially coherent light by a diaphragm of the illumination system. In addition, by changing the numerical aperture of the objective optical system, the incoherent light power can be changed even to partially coherent light to form an image. In general, it is appropriate to observe the illumination stop with an objective optical system having a numerical aperture of about 0.9.

[0011] However, as long as the basics of the optical system and the combination of the optical system and the electronic device are the contrast discrimination ability of the naked eye, the limitation is in the eyes. For example, the contrast discrimination ability of the eyes is the background. Weber'Fechner's law, where L According to A LZL = —Constant (0.01 to 0.02). However, the background light L is established within the range of 3 to 3 × 10 ³ cd Zm ² .

[0012] Further, the resolution of the optical system is a radial force ^ .61 called the Airy disk, which is the first dark ring of the point image by the circular aperture, that is, the two-dimensional Fourier transform of the aperture function by Fraunhofer diffraction. There is a bright circle in the center given by λ ΖΝΑ, which contains about 84% of the light energy. Similarly, the square opening extends along a radial line perpendicular to the side, and the diffracted light spreads in a concentric ring around that. A square opening extends along a radial line perpendicular to the side. Objects smaller than these diffract to this size and spread, and the contrast is significantly lower than real objects. The detection limit of the difference in contrast with the background light is determined by the limit of the eye contrast discrimination ability.

[0013] On the other hand, in recent years, measurement using electronic images has spread, and optical systems are often considered to be part of optical sensors and handle optical signals. According to this idea, the depth, resolution, wavelength band, etc. of the image connected by the optical system can be analyzed as a signal similar to an electromagnetic wave. In addition, the contrast is reduced by background light (for example, imaging characteristics of the optical system, stray light, scattered light), self-luminescence of the optical material and sample holding material, fluorescence, and imaging system noise. . It is now possible to design these comprehensively.

[0014] In order to detect a low-contrast object or an independent minute object, the quantum efficiency of the image sensor is increased, the imaging time is lengthened, or a plurality of images are superimposed to detect the difference in contrast between the optical system and The functions of the image sensor are integrated. Therefore, by reaching the limits of each of them, the limits of the naked eye can be approached or exceeded.

[0015] What is important in developing this logic is the Fourier imaging theory, in which the spatial frequency characteristic of the optical system is made the same as the frequency characteristic of the electric signal transmission line, and the optical signal is Fourier-squeezed. Based on Fourier imaging theory (e.g., Non-Patent Document 2) handled in the petal space, the relationship between the image from the sample passing through the optical system is expressed as a transfer function, and the spatial frequency spectrum of the optical signal Is determined by the transfer function, and is divided into a specific spatial frequency region that is transmitted and a spatial frequency region that is not transmitted. The theoretical boundary is determined by the numerical aperture of the objective lens. For example, in a dry system, the incoherent light is 2λλ, the coherent light is ΝΑΖλ, and the intermediate partial coherent light is determined by the numerical aperture. . [0016] There are the following two methods as methods that exceed this theoretical limit. The basic principle of both is the same, and there is a means for modulating the spatial frequency between the observation object and the imaging optical system. By providing and changing the phase, information on the phase difference that is normally too low to be detected as a contrast difference is reflected in the image as a phase modulation spectrum that includes high-frequency spatial spectral components. Demodulate and synthesize with processing.

[0017] The light modulated by the diffraction grating is demodulated and imaged after passing through the optical system (for example, Patent Document 2 and Non-Patent Document 3), or the light having a phase different by 120 degrees on the diffraction grating. After modulation and passing through the optical system, it is possible to easily demodulate (for example, Patent Document 3 and Non-Patent Document 4), or multiple images captured through the optical system by preparing a spatial modulation pattern. A method of processing and synthesizing has been proposed (for example, Patent Document 4).

[0018] However, as long as a natural biological specimen under a microscope is observed and measured, a wide variety of light amplitudes and phases by various polymers contained therein are detected as contrast differences. The Fourier imaging theory that models physical phenomena has no generality.

[0019] An actual sample is placed on a slide glass, and then encapsulated material is applied and a cover glass is placed on the sample. In current observations, the depth of focus is set to a thickness that allows only a few layers of the sample to form an image!

[0020] However, it is also an important condition that the optical information contained in the sample can be detected as much as possible. If only a part of the layers was observed, information such as signs of important diseases was observed. There is a possibility of dropping. Therefore, the depth of the sample does not affect the observation image due to the difference in contrast due to dust adhering to the surface, dust contained in each layer of the encapsulating material, non-uniformity of the encapsulating material layer, etc. It is also desirable to capture as much material as possible contained in the sample as an image.

[0021] Therefore, the illumination that they can be is incoherent light or partially coherent light. Among them, it is partial coherent light that has an adequate depth of focus and an image of the entire visible light range.

However, since partial coherent light is not linearly imaged, an image of a powerful Fourier component that does not exist in the object appears. This requires analysis by Fourier imaging theory, and the object force cannot define a transfer function to an image via an optical system. However, for example, low The contrast of the trust sample decreases when the background light is brightened. However, the linearity is approximately maintained, so that it can be observed by compensating the decrease with an image sensor.

[0023] As one method, it is said that a transparent and irregularly reflected one or a phase difference larger than 8Zλ or 4Ζλ can be detected even at a low contrast if the microscope has a sufficiently uniform illumination system and a deep focal depth. Yes. As an example, the illumination system and objective system are made telecentric, and the illumination and iris diaphragm are made variable.ΝΑ A variable iris diaphragm objective lens (for example, Patent Document 5), the iris diaphragm is sufficiently in the range of about 0.6 to 0.05. By narrowing down, it is possible to detect large phase changes and transparent and irregular reflections.

[0024] Furthermore, by imaging in a linear sensitivity region in combination with a high-resolution image sensor, the contrast range Δ LZL = constant (0.01 to 0.02) that can be identified by the eyes according to Weber'Fechner's law is set. There are attempts to exceed. It is desirable to have a comprehensive design and signal processing that can improve these performances, and to make the device more suitable for electronic measurement.

[0025] In order to separate the Airy disk and its periphery, it is necessary to separate the light spot and the adjacent point. One of the methods is a graph theory map painting method that separates each country with the smallest number of colors, such as V, where countries adjacent to each other on the world map must be the same color. This is the most famous problem in graph theory, and it has been solved so far! This is based on the hypothesis that any map drawn on the surface of a plane or sphere can be color-coded so that neighboring countries do not have the same color if there are four colors (for example, Non-Patent Document 6). After that, it was proved that it can be painted in four colors with the support of a computer (for example, Non-Patent Document 7). However, it is still unreasonable to be solved elegantly using graph theory in the traditional proof method of mathematics.

Patent Document 1: JP 2002-182119 A (Fig. 2)

Patent Document 2: Japanese Patent No. 3066874 (Fig. 1)

Patent Document 3: Japanese Patent Laid-Open No. 11 242189 (Fig. 1)

Patent Document 4: Japanese Unexamined Patent Publication No. 2001-235316 (Fig. 1)

Patent Document 5: Japanese Patent Laid-Open No. 2003-028460 (FIGS. 2 and 5)

Non-Patent Document 1: E.Hect: "Optics Third Edition", ADDISON WESLEY, pp.453-465, Fi gurel0.30, Figurel0.24, 1998. (Fig. 1, 2)

Non-Patent Document 2: S.INOUE: "VIDEO MICROSCOPY The Fundamentals", Plenum, 1 997.

Non-Patent Document 3: W. Goodman: "Introduction to Fourier Optics", McGraw-Hill, 1986. Non-Patent Document 4: W 丄 ukosz: "Optical System with Resolving Powers Exceeding the CI assical Limit. Π", J. Opt. Soc. Am. 57, pp.932— 941, July 1967.

Patent Document 5: D. Mendlovic et al: One-dimensional superresolution optical system for temporally restricted objects ", J. Applied Optics, Vol.36, pp.2353- 2359, April 1

997.

Non-Patent Document 6: F. Harary: "GRAPH THEORY", Addison- Wesley, 1971

Non-Patent Document 7: K.APPEL, W.HAKEN: "THE SOLUTION OF THE FOUR-COLO

R-MAP PROBLEM ", Sci. Amer. 237 no.4 pp.108-121 1977.

Disclosure of the invention

Problems to be solved by the invention

[0026] A general microscope is an illumination system and an observation system for incoherent light, in which the conditions of high, resolution, depth, and depth necessary to detect all optical information contained in a sample are balanced. It is difficult to achieve the function.

[0027] In order to obtain a high resolution, it is achieved by observing an image with incoherent light. However, the depth of an image formed by using incoherent light is reduced. In addition, deep depth can be achieved by observing images with coherent light. However, although coherent light is generally monochromatic and has a deep depth, for example, in observation and measurement of a biological sample or liquid crystal enclosed between a slide glass and a cover glass, dust or dirt on the sample surface or the glass surface can be obtained. Damage detected along with the sample image.

[0028] Partially coherent light is in the middle, and an image with moderately high resolution and deep depth can be observed. However, partially coherent light is non-linear based on Fourier imaging theory, but remains linear in low contrast samples. In addition, partial coherent light has a lower resolution but deeper depth than incoherent light.

An object of the present invention is to effectively use the resolution of the illumination system and the objective optical system and the image sensor. It is another object of the present invention to provide a microscope imaging apparatus and method for processing and synthesizing image information with contrast density and obtaining a bright image with high resolution and deep depth.

Means for solving the problem

[0030] A microscope imaging apparatus according to the present invention includes an illumination optical system for illuminating a sample, an observation optical system for observing a sample illuminated by the illumination optical system, and an image of the sample observed by the observation optical system. The illumination optical system is provided at a position where a uniform illumination image is formed, uniformly illuminates the entire surface of the sample, and provides coherent light or partially coherent light. An image switching unit configured to illuminate the sample a plurality of times for each illumination group, and the imaging system composes an image corresponding to the sample illuminated by the illumination group to form one image. And the illumination group includes a single bright part or a plurality of bright parts that are not adjacent to each other, and a dark part surrounding the bright part, and the sample is covered by all the bright parts of the illumination group. The entire surface of the lamp is illuminated .

[0031] The microscope imaging method according to the present invention includes an illumination step for illuminating a sample, an observation step for observing the sample illuminated by the illumination step, and an imaging for acquiring an image of the sample observed by the observation step. And the illumination step uniformly illuminates the entire surface of the sample at a position where a uniform illumination image is formed, and the coherent light or the partial coherent light is emitted for each illumination group. An illumination switching step of illuminating the sample a plurality of times, and the imaging step includes an image synthesis step of composing an image corresponding to the sample illuminated by the illumination group to form one image, The illumination group has one bright part or a plurality of bright parts that are not adjacent to each other, and a dark part surrounding the bright part, and the entire surface of the sample is illuminated by all the bright parts of the illumination group. It is characterized by being.

The invention's effect

According to the present invention, light is uniformly illuminated on the entire surface of the sample at a position where a uniform illumination image is formed, and the coherent light or the partial coherent light is illuminated a plurality of times for each illumination group. Then, an image corresponding to the sample illuminated by the illumination group is synthesized to form one image. In this case, the illumination group consists of one bright part or a plurality of bright parts that are not adjacent to each other (also referred to as “light spots” in this specification) and a dark part that surrounds the bright part (in this specification, “dark part”). Also referred to as “point”). And the entire surface of the sample is illuminated by all bright portions of the illumination group.

[0033] With this, high-resolution images can be obtained for incoherent light, and partially coherent light is divided into a light spot connected to the sample surface and a dark spot around it to make the light spot independent. In this way, by detecting partially coherent light or coherent light that also transmits or reflects the sample force, and collects and synthesizes multiple images of illuminated light spots, High resolution images can be acquired.

[0034] The illumination is switched through a liquid crystal shirt array, a reflective micromirror array, a reflective liquid crystal array, or a movable / rotatable pinhole array or a mosaic mirror array. As will be described later, the illumination switching is performed. It is preferable that the number of times of lighting is 4, 9, or 16.

[0035] When imaging information contained in a sample with high resolution, deep depth, and high resolution, the microscope forms an image via an imaging optical system that collects the light of the sample irradiated from the illumination optical system. The purpose of this is to perform the image properly with high resolution and depth and depth.

[0036] Observation of a sample 'imaging is to observe and image an image of light transmitted through or reflected from the sample. These images include high-contrast images and low-contrast images, and biological samples, polymer materials, etc. are low-contrast images. In particular, because biological sample images have low contrast, transmitted light that is easy to detect is used, and a portion of the light irradiated on the sample is evenly distributed on the image plane and does not contain sample information. It is necessary to detect an image with low contrast buried in the background light.

[0037] The illumination includes incoherent light, partially coherent light, or coherent light. Incoherent light is an illumination of a collection of light of various wavelengths and phases, where the entire aperture of the imaging lens is illuminated uniformly by diffused light or condenser illumination. Coherent light is illumination of light of a certain wavelength and phase, and is approximately when the condenser is narrowed down and brought close to a point light source. Partially coherent light is an intermediate illumination and there is no linear relationship between the object and the image. However, if the contrast of an object is sufficiently low, such as a biological sample in microscopic observation, it becomes approximately linear and the transfer function can be defined.

[0038] In order to observe and image a sample with high resolution, illumination light serving as a detection probe and it There is an objective optical system that captures images as images. When the magnification is constant, parameters for determining the image include resolution and depth of focus parameters. About the illumination light,

(1) The sample can be illuminated uniformly;

(2) Illumination can be performed with a stop suitable for the numerical aperture of the objective optical system. For objective optics,

(1) Magnification suitable for imaging,

(2) image brightness,

(3) The numerical aperture can be selected according to the properties of the sample.

A microscope equipped with these illumination system and objective optical system is desired.

[0039] The technology that has been used in conventional confocal microscopes illuminates the sample while moving the light beam through the pinhole to the sample. Also, the scanning beam generated by oscillating a polygonal rotating mirror or mirror is used for the sample. I have been shooting images. However, since the light beam passing through the pinhole is irradiated onto the sample continuously on the scanning line, the coherent light connects the predetermined light spots, but the partially coherent light diffracts according to Fourier imaging theory. Light spreads in a non-linear manner and discontinuously illuminates around the light spot. For example, light energy spreads discontinuously in a circular shape in a circular shape, and light energy spreads unluckily in all directions along each side.

[0040] In the broadening of the light spectrum due to this diffraction, the bright part is the 0th order diffracted light, the 1st order diffracted light, the 2nd order diffracted light, ..., the dark part is the 0th order dark part, the 1st order dark part, the 2nd order This is called the dark part, as shown in Figure 1. The image is the image shown in Figure 2A for a circular bin Honoré, and the image shown in Figure 2B for a square pinhole.

For partially coherent light, nonlinear phenomena such as edge enhancement appear on the image plane due to continuous interference of these lights. In order to avoid strong interference, the map map coloring problem is applied to the diffraction 0th order diffracted light part called Airy's ring, which is the main component of the light amplitude, and the 0th order ring part spreading around it. By surrounding the surrounding illumination as a dark spot and enclosing it as a dark spot, the contrast of the emitted light is emphasized as shown in the graph of Fig. 1.

[0042] In addition, the light irradiated to the sample is absorbed at the light spot and is secondarily excited to affect the surrounding points, or the reflected light may indirectly illuminate the surrounding points. . These shadows In order to reduce reverberation, a dark spot should be provided around the camera, so that mutual indirect illumination due to the light spot is removed so that images can be taken.

[0043] The world map coloring method based on this graph theory is "a method of solving the minimum number of colors required to paint a map" and separates the first term by dark spots. To color-enclose the surrounding area with one dark spot, it is possible to paint with a minimum of four colors (four points). Therefore, in order to illuminate and image with multiple unit light spots, it is possible to illuminate and image all points by changing the pattern at least four times.

[0044] Further, in order to separate to the second term by dark spots, that is, color-coded so that the periphery is surrounded by two dark spots, a minimum of nine colors (9 points) can be used. Therefore, to illuminate and image with multiple unit light spots, if the pattern is changed nine times, all points can be illuminated and imaged. In addition, it is possible to separate at least 16 colors (16 points) to separate up to the 3rd term at the time, that is, to color the surrounding area at three points. Therefore, in order to illuminate and image with multiple unit light spots, if the pattern is changed 16 times, all points can be illuminated and imaged. Thus, in general, about 84% of the light energy is included up to the dark part of the first-order term (Airy circle, corner), and about 92% of the light is included up to the dark part of the second-order term. Therefore, a high transfer function can be achieved by separating the third-order terms and removing the nonlinear components to synthesize the light spot.

[0045] Light that is transmitted or reflected by the sample force illuminated by a uniform independent unit multi-light spot is condensed by a sample surface telecentric optical system equipped with an NA variable iris diaphragm to obtain an image surface telecentric second objective. When projecting onto the imaging surface with a lens, the light spot causes a Fraunhofer diffraction phenomenon regardless of whether it is incoherent, partially coherent, or coherent. The effect of this phenomenon is noticeable for partially coherent light.

[0046] Further, coherent light has a single color and is deep and deep. However, partially coherent light has moderate depth and high resolution even if it is affected by diffraction phenomenon. This is excellent for reading out all image information contained in a thick sample with a microscope. For example, if the depth is too deep, dust attached to the surface of the optical system or the surface of the cover glass may be detected, or spots due to the mounting medium between the cover glass and the sample may be detected. Therefore, an optical system and image processing that sufficiently detect the image information contained in the sample and remove those unrelated to the sample are necessary. [0047] The relationship between depth and resolution is summarized in the following table.

[0048] [Table 1]

In addition, the imaging performance of a microscope is expressed in terms of an optical transfer function (OTF), which can be applied to an illumination system and an observation system. The present invention is a kind of super-resolution technique and is expressed by a comprehensive transfer function including an imaging system. The relationship between contrast and spatial frequency is as shown in the graph in Fig. 3.

[0049] As already described, even in force imaging in which partial coherent light illumination is enhanced by surrounding a transfer function lower than 2ΝΑΖλ with a dark portion around it, a plurality of images taken in conjunction with this are captured. It is possible to acquire high resolution images at a deeper depth by detecting and extracting only the Airy light spots included in the image and combining those light spots into a single image. .

[0050] By matching the unit multi-light spot of the illumination and the unit multi-light spot of the captured image on a one-to-one basis, the light spot can be detected with sufficient contrast, and a high transmission part or reflection part This reduces the indirect illumination effect on the surrounding pixels and the self-luminous phenomenon such as fluorescence, that is, background light or noise).

[0051] By imaging by linking the checkered pattern illumination method in illumination or the checkered pattern image capturing method in function, the imaging element has the highest video frequency when capturing a checkered pattern image. Get higher. The signal of the imaging system at this time repeats the highest signal value and the lowest signal value. This allows the image handling system to work under the most severe conditions. On the other hand, the light spot and the dark spot (reference value) can always be compared, and the accumulation effect of the video signal in the amplifier circuit and the transmission line is the lowest. A lower storage effect means the highest resolution. In addition, since the main component of the video signal is the AC component with the highest frequency, amplification and signal processing (for example, detection by a lock-in amplifier method, AGC: Automatic Gain Control, and multi-image synthesis processing) are performed. It becomes easy. Brief Description of Drawings

[0052] FIG. 1 is a graph showing the spread of light by diffraction.

FIG. 2 is a diagram showing an image showing a circular bin hole and an image showing a square pinhole.

FIG. 3 is a graph showing the relationship between contrast and spatial frequency.

FIG. 4 is a diagram showing a first embodiment of a microscope imaging apparatus according to the present invention.

FIG. 5 is a diagram showing a second embodiment of the microscope imaging apparatus according to the present invention.

FIG. 6 is a diagram showing a third embodiment of the microscope imaging apparatus according to the present invention.

FIG. 7 is a diagram for explaining an example of a pattern of an illumination group when a light spot is surrounded by one dark spot.

FIG. 8 is a diagram for explaining an example of a pattern of an illumination group when a light spot is surrounded by two dark spots.

FIG. 9 is a diagram for explaining an example of an illumination group pattern when a light spot is surrounded by three dark spots.

Explanation of symbols

[0053] 1 light source

2 First collector lens

3 Optical integrator

4 Second collector lens

5-unit multi-point light source

6 Condenser optics

7 Observation optical system

8 Imaging unit

9 samples

10 Calculator

11 Beam splitter

BEST MODE FOR CARRYING OUT THE INVENTION

An embodiment of a microscope imaging apparatus and method according to the present invention will be described with reference to the drawings.

In the drawings, the same elements are denoted by the same reference numerals. Fouri with detailed optical system In the analysis based on er imaging theory, according to Non-Patent Document 2 described above, only the main part will be explained as analogizable.

FIG. 4 is a diagram showing a first embodiment of a microscope imaging apparatus according to the present invention. In this case, the microscope imaging apparatus according to the present invention is realized in a transmission illumination microscope based on Fourier imaging theory, and includes a light source 1, a first collector lens 2, an optical integrator 3, and a second collector lens 4. A unit multi-point light source 5, a condenser optical system 6, an observation optical system 7, and an imaging unit 8.

[0056] As the light source 1, a tungsten lamp, a xenon lamp, a laser light source, or the like can be used. The first collector lens 2 fluoresces the light from the light source 1 to provide uniform and parallel illumination. The light integrator 3 makes the light from the first collector lens 2 more uniform. The second collector lens 4 images the light from the optical integrator 3.

The unit multi-light spot light source 5 is composed of a pinhole array or a liquid crystal shirt array provided at a position where a uniform illumination image is formed. In particular, in the case of a liquid crystal shirt arrangement, the size of the light spot, the size of the dark spot, and the size of the dark area can be appropriately set, so that illumination and imaging conditions suitable for the sample can be set. Thereby, an image with high resolution and deep depth can be obtained. The condenser optical system 6 irradiates the sample 9 by appropriately reducing the illumination of the unit multi-light spot transmitted from the unit multi-light spot light source 5. The observation optical system 8 is composed of, for example, an objective optical system with an NA variable iris diaphragm, and forms an image using light that passes through the sample 9 or is reflected from the sample 9 as incoherent light, partially coherent light, or coherent light. The imaging unit 8 detects the imaged light as an image.

The unit multi-light spot light source 5, the condenser optical system 6, the observation optical system 7, and the imaging unit 8 are controlled and interlocked by the calculation unit 10, and the calculation unit 10 stores the image input from the imaging unit 8. , Process and synthesize and transmit to the outside. Incoherent light uniformly illuminates the entire surface. Partially coherent light or coherent light has a light spot composed of a pinhole array or a transmissive liquid crystal shutter array, and the dark area around it blocks the equivalent unit area. In this way, the sample is illuminated with the light of unit multi-point light. As a result, the diffracted light of the light spot spreads to the dark part that is not illuminated. The light energy is limited to a few percent of the bright part of Airy. In addition, the image connected as incoherent light captures it. The The image combined as the partial coherent light or the coherent light is divided into a plurality of images, and the number of the illuminated images is picked up, input to the calculation unit 10, and processed and synthesized to obtain the entire image.

In order to surround the light spot with a dark spot, a map map coloring problem of a graph is applied. For example, to surround a light spot with one dark spot, at least four colors are required. With four colors, one light spot can be surrounded with a dark spot. As a result, the 0th order dark part and the 1st order bright part of the main nonlinear diffraction can be removed.

[0060] In addition, in order to surround a light spot with two dark spots, at least nine colors are required. With nine colors, one light spot can be surrounded. In other words, nine images are taken, these light spots are processed, combined and taken. As a result, the non-linear main diffraction zero-order dark portion, first-order bright portion, first-order dark portion, and second-order bright portion can be removed.

[0061] Further, in order to enclose a light spot with three dark parts, at least 16 colors are required. With 16 colors, one light spot can be enclosed. That is, 16 images are taken, and these light spots are processed and combined to take an image. As a result, the non-linear main spectrum, ie, the 0th order dark part, the 1st order bright part, the 1st order dark part, and the 2nd order bright part can be removed. This eliminates most of the unwanted nonlinear components.

[0062] By a checkered pattern illumination method or a checkered pattern imaging method, which will be described later, the calculation unit 10 performs an illumination system (unit multi-light spot light source 5 and condenser optical system 6) and an imaging system (observation optical system 7). In addition, the imaging unit 8 can reduce the accumulation effect of the amplifier circuit and the transmission line, and always compare it with the reference value (dark spot). Since the main component of the video signal is the AC component of the highest frequency, amplification and signal processing (for example, detection by the lock-in amplifier method, AGC: Automatic Gain Control, (Combining process) can be easily performed.

FIG. 5 is a diagram showing a second embodiment of the microscope imaging apparatus according to the present invention. In this case, the microscope imaging apparatus according to the present invention is realized in a reflection type illumination microscope based on Fourier imaging theory, and the unit multi-light spot light source 5 is connected to a reflection type micromirror array, a reflection type liquid crystal array, or moving and rotating. It is composed of a free mosaic mirror array, which enables illumination with higher contrast and makes the captured image clearer. This microscope imaging device The second collector lens 4 also reflects a part of the incident light to the unit multi-point light source 5, and reflects the other part of the incident light from the second collector lens 4 units and the unit multi-point. A beam splitter 11 that transmits all of the light incident from the light source 5 is further provided.

FIG. 6 is a diagram showing a third embodiment of the microscope imaging apparatus according to the present invention. In this case, the microscope imaging apparatus according to the present invention is realized in a reflection type illumination microscope based on Fourier imaging theory, and a pinhole in which a unit multi-light spot light source 5 is provided at the position of uniform illumination formed. It also constitutes the alignment or transmission type liquid crystal shirt alignment force. The beam splitter 11 is disposed in the observation optical system 7 and reflects part of the light incident from the unit multi-light spot light source 5 toward the sample 9 and enters from the unit multi-light spot light source 5. Transmits all other incident light from other parts of light and sample 9.

FIG. 7 is a diagram for explaining an example of a pattern of an illumination group in a case where a bright part (light spot) is surrounded by one dark part (dark spot). As already explained, in order to illuminate and image with multiple unit light spots, it is possible to illuminate and image all points by changing the pattern at least four times, so the illumination of 4 patterns of bright parts al to a4 If there is a group (Fig. 7A), the bright parts are formed in order from al to a4 patterns (see Figs. 7B to 7E).

FIG. 8 is a diagram for explaining an example of an illumination group pattern when a bright part (light spot) is surrounded by two dark parts (dark spots). As already explained, in order to illuminate and image with multiple unit light spots, it is possible to illuminate and image all points by changing the pattern a minimum of 9 times, so the illumination of 9 patterns of bright part bl to b9 It is sufficient to have a group (Fig. 8A). First, the bright part is composed of the bl pattern, and finally, the bright part is composed of the b9 pattern (see Figures 8B and 8C).

FIG. 9 is a diagram for explaining an example of a pattern of an illumination group when a bright part (light spot) is surrounded by three dark parts (dark spots). As already explained, in order to illuminate and image with multiple unit light spots, it is possible to illuminate and image all points by changing the pattern at least 16 times, so the illumination group of 16 patterns of bright parts cl to cl6 (Figure 9A) First, the bright part is composed of cl pattern, and finally the bright part is composed of cl6 pattern (see Figures 9B and 9C).

[0068] The present invention is not limited to the above-described embodiment, and many changes and modifications are possible.

For example, the illumination optical system and the observation optical system have a configuration other than the configuration shown in the above embodiment. Can be made. Further, the illumination group can be a pattern other than the pattern shown in the above embodiment, and the number of times of illumination of the coherent light or the partial coherent light can be a number other than 4, 9, 16 times. .

Claims

The scope of the claims

[1] An illumination optical system that uniformly illuminates the sample;

An observation optical system for observing the sample illuminated by the illumination optical system;

An imaging system for acquiring an image of a sample observed by the observation optical system, and the illumination optical system,

Illumination switching means that is provided at a position where an image of uniform illumination is formed, uniformly illuminates the entire surface of the sample, and irradiates the sample with the coherent light or partial coherent light multiple times for each illumination group. Have

The imaging system is

The image corresponding to the sample illuminated by the illumination group is synthesized, and image synthesis means for configuring one image is provided.

The illumination group has one bright part or a plurality of bright parts that are not adjacent to each other and a dark part surrounding the bright part, and the entire surface of the sample is illuminated by all the bright parts of the illumination group. A microscope imaging apparatus characterized by the above.

2. The microscope imaging according to claim 1, wherein the illumination switching means has a liquid crystal shirt array, a reflective micromirror array, a reflective liquid crystal array, or a movable / rotatable pinhole array or a mosaic mirror array. apparatus.

[3] The microscope imaging apparatus according to [1] or [2], wherein the number of illuminations of the illumination switching means is 4, 9, or 16.

[4] an illumination step for illuminating the sample;

An observing step of observing the sample illuminated by the illuminating step; and an imaging step of acquiring an image of the sample observed by the observing step.

There is an illumination switching step that uniformly illuminates the entire surface of the sample at a position where a uniform illumination image is formed, and irradiates the sample multiple times for each illumination group with coherent light or partial coherent light. And

The imaging step includes An image composing step of composing an image corresponding to the sample illuminated by the illumination group to form one image;

The illumination group has one bright part or a plurality of bright parts not adjacent to each other and a dark part surrounding the bright part, and the entire surface of the sample is illuminated by all the bright parts of the illumination group. A microscope imaging method characterized by the above.

5. The illumination switching step is performed through a liquid crystal shirt array, a reflective micromirror array, a reflective liquid crystal array, or a movable / rotatable pinhole array or a mosaic mirror array. Microscopic imaging method.

[6] The microscope imaging method according to claim 4 or 5, wherein the number of times of illumination in the illumination switching step is set to 4, 9, or 16.