JP2023003158A - Fluorescence imaging apparatus - Google Patents

Abstract

To realize a fluorescence imaging apparatus that achieves both high resolution and a wide visual field and has high versatility.SOLUTION: A fluorescence imaging apparatus comprises: a telecentric optical system lens 10; an imaging device 20; a fluorescent filter 61; and image processing means. The image processing means can capture image data of an object to be measured picked up by the imaging device through the telecentric optical system lens, and based on the captured image data, acquire an observation image of the entire visual field of the object to be measured and an analysis image for spatially resolving two points adjacent on a micron order or less in a predetermined area in the visual field of the object to be measured.SELECTED DRAWING: Figure 1

Description

Patent Act Article 30, Paragraph 2 application filed (Trans-scale-scope to find rare cellular activity in sub-million cells) Publication date: June 30, 2020 [Publications] (Exploring rare cellular activity in (more than one million cells by a trans-scale-scope) Issue date: March 2, 2021

The present disclosure relates to a fluorescence imaging apparatus that measures the fluorescence phenomenon of a measurement object such as living cells, and in particular, enables overall observation with a wide field of view and high-resolution magnified observation of each part of the measurement object included in the field of view. The present invention relates to a fluorescence imaging apparatus that is compatible and highly versatile.

A fluorescence microscope is conventionally used for fluorescence observation of living cells. This fluorescence microscope has an illumination optical system that excites the fluorescence emission of the object to be measured, an imaging lens system that optically magnifies the object to be measured, and an enlarged image of the object to be measured obtained by the imaging lens system. It consists of an imaging device that acquires data.

Spatial resolution in a fluorescence microscope is determined by the numerical aperture (NA) and lens magnification of the imaging lens system and the pixel size of the imaging device. On the other hand, the size of the observation field of view is determined by the field number and lens magnification of the imaging lens system, and the overall size of the imaging device.

In a fluorescence microscope, there is a trade-off relationship between spatial resolution and the width of the field of view. If a high-resolution imaging lens system is used, the field of view becomes narrower, and conversely, if a wide field of view is secured, the resolution decreases. As a currently used fluorescence microscope, for example, when a 2K-CMOS camera with a pixel size of 6.5 μm is used as an imaging device, the resolution is 300 nm at a wavelength of 500 nm when using a high magnification lens of 40 times and NA 0.95. can be secured, but the field of view remains at 450 μm. Conversely, if a low-magnification lens with a lens magnification of 2× and NA of 0.10 is used, the resolution is 6.5 μm, but the field of view is as wide as 9 mm.

In recent years, with the aim of achieving both high resolution and a wide field of view, the University of Strathclyde in the United Kingdom created a giant lens called Mesolens, which has a wide field of view of 6 mm x 6 mm and a high resolution of 0.7 μm fluorescence imaging microscope. has been proposed (see Non-Patent Document 1). In addition, Tsinghua University in China has proposed an imaging device called RUSH that achieves a field of view of 12 mm × 10 mm and a high resolution of 0.92 μm using a huge objective lens and an arrayed CMOS camera (non-patent Reference 2).

McConnell, G and Amos, W.B. “Application of the Mesolens for subcellular resolution imaging of intact larval and whole adult Drosophila Imaging intracellular fluorescent proteins at nanometer resolution” Journal of Microscopy, Vol. 270, Issue 2 2018, pp. 252-258 Zheng G el al. "0.5 gigapixel microscopy using a flatbed scanner" Biomedical Optics Express, Vol. 5, Issue 1 2014

According to the conventional observation methods described above, fluorescence imaging observation can be performed with high resolution and a wide field of view. However, in any of the above methods, since the lenses are huge and expensive, they are limited to being used by the research institutes that made the prototypes, and lack versatility such as commercialization due to problems of cost and mass production. Met.

SUMMARY OF THE INVENTION Accordingly, it is an object of the present application to solve the above-described conventional problems and realize a fluorescence imaging apparatus that achieves both high resolution and a wide field of view and is highly versatile.

In order to solve the above problems, the fluorescence imaging apparatus disclosed in the present application includes a telecentric optical system lens, an imaging device, a fluorescence filter, and image processing means, wherein the image processing means uses the telecentric optical system lens. image data of the object to be measured captured by the imaging device through the camera, and an observation image of the entire field of view of the object to be measured and a predetermined region within the field of view of the object to be measured based on the image data taken in It is characterized in that it is possible to acquire an analysis image that spatially resolves two adjacent points on the micron order or less in .

The fluorescence imaging apparatus disclosed in the present application can acquire a magnified image of a desired region of the measurement object by the image processing means from the image data captured by the imaging device through the telecentric optical system lens. Therefore, in addition to the entire image with a wide viewing angle, fluorescence imaging observation can be performed based on an analysis image that is substantially uniform and has high resolution in any part of the field of view.

FIG. 1 is a diagram showing the overall configuration of a fluorescence imaging apparatus according to this embodiment. FIG. 2 is an image diagram for explaining irradiation of irradiation light of the fluorescence imaging apparatus according to this embodiment. FIG. 3 is a diagram showing profiles from analysis images of 200 nm beads in the central and peripheral portions of the field of view of the fluorescence imaging apparatus according to this embodiment. FIG. 4 is a diagram showing an example of observation of living cells with the fluorescence imaging apparatus according to this embodiment. FIG. 4 shows an overall image of the object to be measured. FIG. 5 is a diagram showing an example of observation of living cells with the fluorescence imaging apparatus according to this embodiment. FIG. 5 shows an enlarged image obtained by image processing of the portion indicated by A in FIG. FIG. 6 is a diagram showing an example of observation of living cells with the fluorescence imaging apparatus according to this embodiment. FIG. 6 shows an enlarged image obtained by image processing of the portion indicated by B in FIG.

The fluorescence imaging apparatus of the present disclosure includes a telecentric optical system lens, an imaging device, a fluorescence filter, and image processing means, and the image processing means is imaged by the imaging device via the telecentric optical system lens. Capture image data of a measurement object, and based on the captured image data, an observation image of the entire field of view of the measurement object and two adjacent points on the order of microns or less in a predetermined region within the field of view of the measurement object. It is possible to obtain an analysis image that spatially resolves the

By doing so, the fluorescence imaging apparatus of the present disclosure acquires an optical image with less distortion obtained by a telecentric optical system lens as image data with high resolution by an imaging device with a large number of pixels and a small pixel size. By using the image processing means, it is possible to obtain a desired analysis image such as an entire image within the field of view of the object to be measured, or a high-resolution image that can spatially resolve any two points adjacent to each other on the order of microns or less. can. Further, by using a telecentric optical system lens that is widely used in an inspection apparatus for industrial products as the optical lens system, it is possible to realize a fluorescence imaging apparatus with high versatility.

In the fluorescence imaging apparatus of the present disclosure, it is preferable that the magnification of the telecentric optical system lens is 5 times or less. By doing so, it is possible to make the most of the telecentric optical system lens feature that it is possible to acquire an optical image with little optical aberration, and the entire area of the captured image acquired by the imaging device can be captured with high resolution. analysis image can be obtained.

Further, it is preferable that the irradiation light with which the object to be measured is irradiated is irradiated with dark field illumination from outside the viewing angle of the telecentric optical system lens. By doing so, the S/N ratio of light emitted from the object to be measured in the captured image is improved, and the object to be measured can be easily irradiated with irradiation light of a desired wavelength (color).

Furthermore, it is preferable that the object to be measured is placed in an environment in which temperature and CO ₂ concentration are controlled. By doing so, it is possible to maintain the condition of a biological material such as a cell, which is an object to be measured.

Furthermore, the fluorescence imaging apparatus disclosed in the present application preferably has a visual field size of 10 mm or more on one side and is capable of acquiring an analysis image that spatially resolves two adjacent points within 3 microns or less. By doing so, it is possible to perform fluorescence observation at a high resolution of one cell level or more in each part as well as the entire image of an object having a certain size or more as a measurement object.

(Embodiment)
An embodiment of a fluorescence imaging apparatus according to the present disclosure will be described below with reference to the drawings.

FIG. 1 is an external perspective view for explaining the configuration of the fluorescence imaging apparatus according to this embodiment.

A fluorescence imaging apparatus 100 shown in FIG. acquired by the imaging device 20. Note that the image data of the object to be measured acquired by the imaging device 20 is displayed as an entire image within the imaging field of view or as an enlarged image in which a portion is enlarged by data processing in an image processing unit (not shown). Then, image data desired by the user can be output.

The telecentric optical system lens 10 is a lens that is set so that the optical axis and the principal ray are parallel. It is used in product inspection equipment, etc. In the fluorescence imaging apparatus 100 disclosed in the present application, focusing on the fact that almost no optical aberration occurs in the peripheral portion of the field of view, which cannot be avoided with a non-telecentric optical system used in a conventional fluorescence microscope. A telecentric optical system lens is used as an optical lens for acquiring image data.

The telecentric optical system lens 10 is used in the fluorescence imaging apparatus 100 according to the present embodiment, as described above, to avoid distortion of an acquired image due to optical aberrations such as curvature of field and distortion in the entire range of the field of view. Therefore, it is preferable that the telecentric optical system lens 10 to be used has a low magnification because it causes less image distortion. Specifically, the lens magnification is preferably 5 times or less, and it is more preferable to use a telecentric optical system lens 10 with a lens magnification of 1 to 2 times. Also from the viewpoint of obtaining an entire image of a wide area of the object to be measured, the telecentric optical system lens 10 used in the fluorescence imaging apparatus according to this embodiment is preferably 5 times or less.

As the telecentric optical system lens 10, a commercially available lens for inspection equipment for industrial products can be used. In particular, a telecentric optical system lens for a macro type inspection apparatus can be preferably used.

The image pickup device 20 can use a general image pickup element such as CMOS or CCD, in which fine image pickup pixels are arranged in a matrix and an image is formed by photoelectric conversion in each pixel. In addition, in the fluorescence imaging apparatus 100 disclosed in the present application, based on the image data acquired by the imaging device 20 via the telecentric optical system lens 10 described above, data processing is performed on the order of microns, that is, two adjacent points at 5 μm or less It is possible to acquire an analysis image having a resolution for spatially resolving the Therefore, the imaging device 20 used needs a sampling period of 2.5 μm or less, which is half of 5 μm, and the pixel size needs to be 2.5 M μm or less, where M is the magnification of the telecentric optical system lens. .

In addition, the pixel size and the number of pixels also affect the size of the imaging area of the imaging device 20 used. In the fluorescence imaging apparatus 100 shown in this embodiment, the telecentric optical system lens 10 for inspection of industrial products can be used as described above. A standardized imaging device 20 of size (diagonal 33 mm) or 35 mm full size (diagonal 44 mm) can be used.

As shown in FIG. 1, irradiation light (excitation light) 36 emitted from irradiation light sources 31 (31a, 31b, 31c) that irradiate predetermined wavelengths (for example, wavelengths of 385 nm, 470 nm, and 525 nm) respectively is a dichroic mirror. 32a, 32b, the first lens 33, the second lens 34, and the mirror 35, the sample 90 to be measured is irradiated with the light.

As the irradiation light source 31, an LED element, a xenon lamp, or the like is used as a light source, and if necessary, a filter is used to realize irradiation light of a desired wavelength.

FIG. 2 is an image diagram showing how the measurement object is irradiated with the irradiation light in the fluorescence imaging apparatus 100 according to the present embodiment.

As shown in FIG. 2, the illumination light 36 is incident from outside the NA (θ=7° as an example) of the telecentric optical system lens 10 at a larger angle α (an angle of 35° as an example). The measurement object 90 is irradiated through the cover glass 72 a of the sample dish 71 .

By irradiating the illumination light as dark field illumination instead of the epi-illumination used in conventional fluorescence microscopes, even if a telecentric optical system lens 10 with a large lens movable range is used, the captured image is acquired without any influence. A high S/N ratio can be realized by improving the contrast of the image data. In addition, by adopting dark field illumination, a mechanism such as a half mirror for irradiating the object to be measured with irradiation light is not required in the imaging lens optical system, and a commercially available telecentric optical system lens 10 can be used as it is. . Further, by irradiating the irradiation light from the outside of the imaging optical system, the irradiation optical system for irradiating the irradiation light 36 from the irradiation light source 31 to the mirror 35 can be replaced with the imaging optical system for acquiring the image data. Since the design can be done separately, modifications such as changing the wavelength of the irradiation light can be easily performed.

Although illustration is omitted, the fluorescence imaging apparatus 100 according to the present embodiment has a separate light source using a white light LED as epi-illumination, and bright-field imaging is performed by irradiation light A ( 1).

A sample as a measurement object 90 is mounted on a mounting table 41 and adjustment mechanisms 42 (42a, 42b, 42c) capable of finely adjusting the position of the mounting table 41 in the X-axis direction, the Y-axis direction, and the Z-axis direction, respectively. is placed on a mounting stage 40 having

Further, the telecentric optical system lens 10 is attached to an elevating mechanism 50 that can move in the vertical direction. Adjusted to bring the desired portion of the object to be measured within the field of view of the lens.

As an arrangement mechanism 60 for the fluorescence filter 61 required for fluorescence imaging observation, a conventionally used filter switching system (reference numeral 62) or a so-called revolver system (reference numeral 63) can be appropriately adopted. In the fluorescence imaging apparatus 100 according to this embodiment, by changing the combination of the wavelength of the irradiation light 36 and the fluorescence filter 61, fluorescence observation in multiple colors becomes possible.

In the fluorescence imaging apparatus 100 of the present embodiment, considering that living cells are used as the measurement object 90, the sample dish 71 containing the measurement object 90 is arranged in the thermostatic chamber 72, and the temperature inside the thermostatic chamber 72 is A gas supply hose 73 is arranged to create a CO ₂ gas atmosphere in the space.

An elevating mechanism 50 for elevating the telecentric optical system lens 10 and the imaging device 20 arranged integrally with the telecentric optical system lens 10 can elevate one side of a post 81 fixed to a support base 82. A mounting stage 40 on which an observation object 90 is mounted and a fluorescence filter 61 for fluorescence observation are also fixed to the same side surface of the column 81 .

FIG. 3 shows profiles analyzed from image data of fluorescent beads captured by the fluorescence imaging apparatus according to this embodiment.

More specifically, first, a plurality of fluorescent beads having a diameter of 200 nm and a center wavelength of 520 nm were prepared as the observation object 90 and randomly dispersed within the field of view of the imaging optical system. An image of the dispersed fluorescent beads was obtained using an LED with a central wavelength of 470 nm as the irradiation light source 31 . The imaging device 20 in the fluorescence imaging apparatus 100 used at this time has 120 million pixels (the number of effective pixels is 13264×9180), the pixel size is 2.2 μm, and the chip size is 29.2 mm×20.2 mm. -H size, and the telecentric optical system lens 10 has a magnification of 2 times. The field size at this time was 14.6 mm×10.1 mm (diagonal 17.8 mm).

From the obtained captured image of the fluorescent beads, the luminescence intensity distribution in the y-axis direction was analyzed for the beads in the central portion of the imaging field and the beads near the corners of the imaging field. FIG. 3(a) shows the data for the beads in the central portion of the imaging field, and FIG. 3(b) shows the data for the beads in the vicinity of the corners of the imaging portion. In each data, the circles a and a' indicate the measured values of the emission intensity at the corresponding positions. The measured values represent the light emission intensity of each pixel of the imaging device 20, and the analysis results are obtained at intervals of 1.1 μm. Luminescence intensity distribution curves obtained by the Gaussian fit method for this measured data are shown as b and b', respectively.

The full width at half maximum (FWHM) of the emission intensity distribution curves shown in FIGS. 3(a) and 3(b) is the portion indicated by the arrow in the figure, and is 2.25 μm at the central portion of the field of view shown in FIG. 3(a). , and 2.28 μm in the peripheral portion of the field of view shown in FIG. From this result, according to the fluorescence imaging apparatus 100 according to the present embodiment, it is possible to acquire an image that can detect fluorescence that is in contact with a distance of several microns in any part of the field of view, and the spatial resolution of the fluorescence imaging apparatus 100 is microns. It has been confirmed that two adjacent points can be resolved below the order.

The FWHM value obtained from the above actual measurement results is the theoretical PSF (=0.12) obtained from NA=0.12 of the telecentric optical system lens used and the emission wavelength λ of the fluorescent beads=520 nm. It was also confirmed that it is close to the theoretical value of 2.17 μm calculated by the FWHM of 50λ/NA).

In addition, although not illustrated, the inventors simultaneously measured the spatial resolution in the plane shown in FIG. Acquisitions were also analyzed for resolution in the Z direction from the fluorescent bead to the lens. Since the telecentric optical system lens 10 with a low NA of NA = 0.12 was used as described above, the depth of focus was much longer than the cross section, and the measured value at the center of the field of view was 62.4 μm, calculated by the Gaussian fit method. It was confirmed that the value was 64.0 μm, the measured value was 69.4 μm, and the calculated value was 64.2 μm near the corner.

From this result, it can be confirmed that the spherical aberration due to the cover glass 72a (for example, 170 μm thick) and the filter 61 having a thickness of 2 mm, which are present between the measurement object 90 and the telecentric optical system lens 10, can be almost ignored. rice field.

From the above study results, it was confirmed that the fluorescence imaging apparatus 100 according to the present embodiment could achieve substantially uniform spatial resolution over the entire field of view.

4 to 6 show analysis results of a mouse brain showing an example of cell observation using the fluorescence imaging apparatus according to this embodiment.

In the fluorescence imaging apparatus 100 according to the present embodiment, multi-color fluorescence imaging of the measurement object 90 is possible by sequentially switching the combination of the irradiation light source 31 and the fluorescence filter 61 at a plurality of wavelengths, particularly at three or more wavelengths. .

FIG. 4 shows a whole image of a centimeter-wide mouse brain sliced into 25 μm-thick slices as an example of multicolor fluorescence imaging observation.

FIG. 5 is an enlarged image of the area (cerebral cortex) shown as region A in FIG. 4 by image processing. It is a figure magnified twice (25 times in total).

FIG. 6 is an enlarged image of the portion (hippocampus) shown as region B in FIG. 4 by image processing. It is a figure enlarged (total of 25 times).

The observation images shown in FIGS. 4 to 6 show the fluorescence intensity of tdTomato expressed in excitatory projection neurons (red), EGFP expressed in inhibitory interneurons (green), and Hoechst33342 bound to nuclear DNA (blue). ing. Note that the three color channels were excited using three LED wavelengths (center wavelength: 525 nm, 470 nm, 385 nm) and the exposure time for each channel was 2 seconds.

From FIG. 4, the analysis image of the entire mouse brain within the field of view of the fluorescence imaging apparatus 100 according to the present embodiment, and from FIGS. 5 and 6, two specific regions (cerebral cortex, hippocampus). It was confirmed that enlarged images can be obtained. In the 25-fold enlarged images shown in FIGS. 5(b) and 6(b), the distribution of single nerve cells and neurites could be observed.

As described above, with the fluorescence imaging apparatus 100 according to the present embodiment, since it is possible to acquire enlarged image data in each portion within the field of view by acquiring image data once, each enlarged image in each region of the measurement object can be acquired. No need to get it in multiple colors. Therefore, three-color image data for multi-color observation of the entire area of the measurement object 90 can be acquired in several seconds. Since such high-speed data acquisition and data analysis are possible, the fluorescence imaging apparatus 100 according to the present embodiment can significantly reduce the image data acquisition time required for conventional fluorescence microscope observation. can be done.

In addition, as described above, since the entire brain slice of centimeter order can be observed at once, whole brain imaging, which has been emphasized in the field of neuroscience in recent years, can be handled at high speed.

Furthermore, the fluorescence imaging apparatus 100 according to the present embodiment has a wide field of view, and by taking advantage of the ability to simultaneously acquire enlarged image data on the order of microns over the entire field of view, various time-lapse observations can be effectively performed. be able to. In particular, the wide-field and high-resolution fluorescence imaging apparatus 100 can Many observations that can only be made are assumed.

As described above, the fluorescence imaging apparatus 100 according to the present embodiment uses an easily available telecentric optical system lens and a high-resolution imaging device to achieve a wide field of view that could not be achieved with conventional fluorescence microscope observation. Therefore, it is possible to realize an observation device capable of fluorescence observation with high resolution.

The fluorescence imaging apparatus disclosed in the present application is expected to contribute to research in various fields including the life science field as a highly versatile fluorescence observation apparatus that achieves both a wide field of view and high resolution.

REFERENCE SIGNS LIST 10 telecentric optical system lens 20 imaging device 61 fluorescence filter 100 fluorescence imaging device

Claims (5)

A telecentric optical system lens, an imaging device, a fluorescence filter, and an image processing means,
The image processing means captures image data of the object to be measured captured by the imaging device through the telecentric optical system lens, and based on the captured image data, produces an observation image of the entire field of view of the object to be measured. , and an analysis image obtained by spatially resolving two adjacent points on the order of microns or less in a predetermined area within the field of view of the object to be measured. 2. The fluorescence imaging apparatus according to claim 1, wherein said telecentric optical system lens has a magnification of 5 times or less. 3. The fluorescence imaging apparatus according to claim 1, wherein the irradiation light applied to the object to be measured is applied by dark field illumination from outside the viewing angle of the telecentric optical system lens. 4. The fluorescence imaging apparatus according to any one of claims 1 to 3, wherein said measurement object is placed in an environment in which temperature and CO ₂ concentration are controlled. 5. The fluorescence imaging apparatus according to any one of claims 1 to 4, wherein one side of the visual field size is 10 mm or more, and an analysis image can be obtained by spatially resolving two adjacent points in 3 microns or less.

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