Autofluorescence Quencher

Abstract

Disclosed is an autofluorescence quenching device that: quenches only autofluorescence without quenching fluorescence that is originally desired to be observed in a tissue section for a short time; does not emit noise in various wavelength regions; is capable of controlling a temperature increase; can be used repeatedly with a simple operation; and can stably quenches autofluorescence. An autofluorescence quenching device for a sample comprises: a sample placement unit; and an irradiation unit that is placed atop the sample placement unit. The sample placement unit is equipped with: a flat plate; one or more sample placement recesses which are provided in the flat plate and in which samples are placed; and a cooling means that cools the sample placement recesses. The irradiation unit is equipped with one or more white LED light-sources that are each placed above the respective sample placement recesses.

Description

[Technical Field]

[0001] The present invention relates to an autofluorescence quenching device for quenching autofluorescence from a biological sample or the like. [Background technology]

[0002] Advances in optical machinery and staining techniques have enabled researchers to delve deeper into the mechanisms underlying biological phenomena, even at the cellular and subcellular levels. To accurately capture the macroscopically visible characteristics of biological organs and tissues, fluorescent imaging techniques using fluorescent probes must take into account phenomena and characteristics such as fluorescence quenching, fluorescence intensity, photobleaching, and fluorescence background. Without a method to quench or mask autofluorescent substances such as lipofuscin derived from living organisms or fluorescence from tissue fixative treatments, it is virtually impossible to consistently capture and image fluorescent staining signals independent of the state of the tissue or cells. Therefore, in the field of life sciences, particularly histology (pathology), there is a strong demand for a technology that can efficiently quench autofluorescence.

[0003] Here, conventional techniques for quenching autofluorescence can be classified into a chemical quenching technique using a dedicated reagent and a physical quenching technique using a light source such as a mercury lamp to irradiate the tissue slice with light.

[0004] Although chemical quenching techniques can eliminate autofluorescence, they have many drawbacks, such as the elimination of the fluorescence that you want to observe, an increase in noise in other wavelength ranges, and high costs depending on the number of samples, and there have not been many published papers using these techniques.

[0005] On the other hand, although physical quenching techniques can eliminate autofluorescence, they require a long time of 4 to 48 hours for quenching, and many drawbacks remain, such as irreversible denaturation of tissue sections due to temperature rise during irradiation, which must be avoided (Non-Patent Document 1). Therefore, there have been few published papers using physical quenching techniques. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Doung H and Han M 2013 A multispectral LED array for the reduction of background autofluorescence in brain tissue. Journal of neuroscience methods, 220: 46-54 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made in view of the above-mentioned circumstances of the prior art, and an object of the present invention is to provide a technology that can quench only the autofluorescence of a tissue section in a short time without quenching the fluorescence that is actually desired to be observed, that does not produce noise even in various wavelength ranges, and that can control temperature rise, that is simple to operate, can be used repeatedly, and is capable of stably quenching autofluorescence. [Means for solving the problem]

[0008] As a result of extensive research, the inventors of the present invention have discovered that by using a white LED illumination as an illumination source, it is possible to quench only the autofluorescence of tissue sections in a short time without quenching the fluorescence that is the intended subject of observation, and that noise does not occur even in various wavelength ranges. Furthermore, they have conceived an autofluorescence quenching device that uses a white LED illumination as an illumination source, can suppress temperature rise, and can be used repeatedly with simple operation, and have completed the present invention.

[0009] That is, the present invention provides the following. (1) An apparatus for quenching autofluorescence of a sample, comprising a sample placement unit and an irradiation unit placed on the sample placement unit, the sample placement unit includes a flat plate, one or more sample placement recesses provided on the flat plate for placing samples therein, and a cooling means for cooling the sample placement recesses; The illumination unit includes one or more white LED lights respectively disposed above each of the sample placement recesses. Autofluorescence quencher. (2) The autofluorescence quenching device according to (1), wherein the cooling means is a heat sink and / or a cooling fan provided below the flat plate. (3) The autofluorescence quenching device according to any one of (1) to (3), wherein the irradiation unit includes a second flat plate, and the white LED illumination is disposed on the second flat plate. (4) The autofluorescence quenching device according to (3), wherein the irradiation unit further comprises a second cooling means. (5) The autofluorescence quenching device according to (4), wherein the second cooling means is a heat sink and / or a cooling fan provided on the second flat plate. (6) The autofluorescence quenching device according to any one of (3) to (5), wherein the irradiation unit is placed on the sample placement unit via a rib, and the rib provides a space between the flat plate and the second flat plate. [Effects of the Invention]

[0010] The present invention provides an autofluorescence quenching device that can quench only the autofluorescence of tissue sections in a short time without quenching the fluorescence that is originally intended to be observed, does not generate noise even in various wavelength ranges, and can control temperature rise, is simple to operate, can be used repeatedly, and can stably quench autofluorescence. [Brief explanation of the drawings]

[0011] [Figure 1] 1 is a schematic perspective view of a preferred embodiment of an autofluorescence quenching device of the present invention. [Figure 2] FIG. 2 is a partial perspective cross-sectional view of the specific example shown in FIG. [Figure 3]FIG. 2 is a schematic perspective view showing the irradiation unit of the specific example shown in FIG. 1 upside down. [Figure 4] FIG. 2 is a schematic perspective view showing a sample placement unit of the specific example shown in FIG. 1. [Figure 5-1] This figure shows the relative amount of quenching time and degree of quenching in each wavelength range in Example 1. It shows the amount of autofluorescence light observed in the mouse cerebral cortex where cellular tissue is present in each wavelength range: 425-475 nm (blue), 500-550 nm (green), 570-620 nm (red), and 650-750 nm (infrared). Zero minutes of light irradiation is set to 100%, and the amount of light in the lumen of blood vessels and other areas where no mouse tissue is present is set to 0%. The values ​​are shown as the average and standard deviation. [Figure 5-2] This figure shows the results of the same experiment as in Figure 5-1, with the maximum values ​​shown. DETAILED DESCRIPTION OF THE INVENTION

[0012] A preferred embodiment of the present invention will now be described with reference to the drawings.

[0013] FIG. 1 is a schematic perspective view of a preferred embodiment of the autofluorescence quenching device of the present invention, FIG. 2 is a partial perspective cross-sectional view of the embodiment shown in FIG. 1, FIG. 3 is a schematic perspective view of the embodiment shown in FIG. 1 with the irradiation unit turned upside down, and FIG. 4 is a schematic perspective view of the embodiment shown in FIG. 1, showing the sample placement unit.

[0014] As shown in FIG. 1, the autofluorescence quenching device of the present invention comprises a sample placement unit 10 and an irradiation unit 12 placed on the sample placement unit 10. As shown in FIG. 4, the sample placement unit 10 comprises a flat plate 103. While the material of the flat plate 103 is not particularly limited, a metal that dissipates heat easily is preferred, such as silver, copper, or aluminum. The upper surface of the flat plate 103 may be mirror-finished to improve light reflection. Alternatively, a material with low heat absorption, such as glass or acrylic, may be used, and heat generation may be suppressed by irradiating light in a liquid. The flat plate 103 has multiple sample placement recesses 107 formed therein. While a single sample placement recess 107 is sufficient, multiple recesses are preferred, as they allow for simultaneous processing of multiple samples. In the illustrated example, the sample placement recess 107 is cylindrical, but the shape is not particularly limited. For example, the recess 107 may be shaped to accommodate a slide glass, culture dish, cover glass, or the like. When the sample placement recess 107 is cylindrical, the size is not particularly limited, but the diameter of the circle is usually about 10 mm to 20 mm, and the depth is about 2 mm to 8 mm.

[0015] A heat sink 102, which serves as a cooling means for the sample placement recess 107, is placed below the flat plate 103, and a fan 101 (see Figure 2) is placed below that. While either the heat sink 102 or the fan 101 alone can be used as the cooling means, it is preferable to have both, as this improves cooling performance. In addition to a heat sink or a fan, a Peltier cooling unit, a cold water chiller unit, etc. can also be used as the cooling means.

[0016] On the other hand, the irradiation unit 12 includes a second plate 108. As shown in FIG. 3, multiple white LED illuminators 106 are disposed on the second plate 108. The material of the second plate 108 is not particularly limited, but a metal that dissipates heat easily is preferred, such as silver, copper, or aluminum. The upper surface of the second plate 108 may be mirror-finished to improve light reflection. The multiple white LED illuminators 106 are disposed corresponding to the multiple sample placement recesses 107, and are positioned so that, when the irradiation unit 12 is placed on the sample placement unit 10, each of the multiple white LED illuminators is disposed directly above each sample placement recess 107. The distance between the lower surface of the white LED illuminator 106 and the bottom surface of the sample placement recess 107 is typically approximately 3 mm to 20 mm, preferably approximately 4 mm to 10 mm. The white LED illuminators 106 are connected to a power source (not shown) and are turned on during use. The intensity of the white LED lighting during use is set appropriately, but is typically around 1,000 to 8,000 lumens, and preferably around 1,500 to 5,000 lumens. The output of the white LED lighting is preferably adjustable between 0 and 1,000 W, preferably between 0 and 500 W, and more preferably between 0 and 400 W, with the output setting resolution preferably in 100 W increments, and even more preferably in 10 W increments. White LED lighting typically emits white light by combining a blue LED with a yellow phosphor, its complementary color. The blue LED preferably has a dominant wavelength (peak wavelength) of around 450 to 520 nm, more preferably 470 to 500 nm.

[0017] Ribs 104 are disposed between the second flat plate 108 and the flat plate 103 of the sample placement unit 10 (see FIG. 3). The ribs 104 function as spacers to provide a distance between the flat plate 103 of the sample placement unit 10 and the second flat plate 108 of the irradiation unit 12, thereby ensuring that the distance between the lower surface of the white LED light 106 and the bottom surface of the sample placement recess 107 is a desired value.

[0018] A second heat sink 109, which is a cooling means, is disposed on the second flat plate 108, and a second fan 110, which is also a cooling means, is disposed on top of that. The irradiation unit 12 does not necessarily need to have a cooling means, but it is preferable to have one since it can further suppress the temperature rise of the sample. Also, while it is possible to have only the heat sink 109 or the fan 110, it is preferable to have both since this improves cooling performance.

[0019] Furthermore, a thermometer probe insertion port 105 may be formed in the flat plate 103, allowing a thermometer probe to be inserted therein to measure the temperature inside the flat plate 103 (close to the temperature of the sample placement recess 107). In this case, the apparatus may be equipped with a control means for controlling the drive current of the irradiation unit 12, a control means for controlling the drive current of the cooling means of the sample placement unit 12, a temperature detection means for detecting the temperature of the sample placement unit 12, a setting means for setting a predetermined temperature threshold value that takes into consideration the temperature detection means, a determination means for determining whether or not an abnormality has occurred based on the threshold value set by the setting means, and a stopping means for automatically stopping the drive current based on the determination by the determination means, thereby preventing the sample from being undesirably heated and damaged.

[0020] During use, a sample, such as a biological tissue slice, is placed in the sample placement recess 107. The type of sample, such as a biological tissue slice, is not particularly limited; any biological species or tissue material may be used as long as it is large enough to fit into the sample placement recess 107. Next, the illumination unit 12 is placed on the sample placement unit 10. This positions each white LED illuminator 106 directly above each sample placement recess 107. In this state, each white LED illuminator 106 is turned on. The illumination time can be set as appropriate, but is typically about 15 minutes to 1 hour. This operation quenches the autofluorescence of the sample. During use, the entire autofluorescence quenching device may be attached to a shaker and irradiated while being shaken. In this case, the sample in the sample placement recess 107 moves relatively within the recess to some extent, which has the advantage of allowing for more uniform illumination of the sample.

[0021] The present invention will be specifically described below based on examples, although the present invention is not limited to the following examples.

[0022] Example 1 All animal experiments were conducted in accordance with the guidelines of the Toho University Animal Care and Use Committee, and procedures were approved by the committee (approval number 19-51-405). Mating pairs of C57 / BL6 mice were purchased from Japan SLC Co., Ltd. and CLEA Japan, Inc. Mice were housed in a breeding colony within the university under controlled conditions of a 12-hour light-dark cycle, 23 ± 2°C, and 55% ± 5% humidity. Brains were collected from 20-30 week-old male mice. Mice were deeply anesthetized with intraperitoneal administration of 50 mg / ml sodium pentobarbital and transcardially perfused with 4% paraformaldehyde in phosphate buffer. The removed brains were postfixed overnight in 4% formaldehyde at 4°C, cryoprotected by infiltration in 30% sucrose solution for two days, mounted in Surgipath (Leica Biosystems), and stored at -80°C until use. Brain sections were prepared as frozen sections at a thickness of 40 μM.

[0023] An autofluorescence quenching device according to one embodiment of the present invention, as shown in Figures 1 to 4, was prepared. The dominant wavelength of the blue LED constituting the white LED light 106 was 475 nm. After washing the tissue section with phosphate buffer, it was transferred using a brush to a sample placement recess 107 filled with the buffer solution. The sections were either not irradiated or irradiated for 15, 30, 45, or 60 minutes. The light irradiation was uniformly performed under conditions of 24 W for each LED. The LED had an intensity of 4022 lumens at 32 W. The sample placement recess 107 had a diameter of 15 mm, a depth of 4 mm, and a distance of 5.5 mm from the LED to the bottom of the sample placement recess 107. During light irradiation, the tissue section was shaken at 60 rpm using a shaker to ensure uniform light irradiation conditions, and the sample placement recess 107 was continuously cooled to maintain a temperature of 37°C or below.

[0024] Fluorescence photographs of stained tissue sections were taken in the cerebral cortex using a Nikon Eclipse Ni microscope equipped with an A1R confocal detection system. The laser wavelengths used for fluorescence excitation were 405 nm, 488 nm, 560 nm, and 640 nm. Fluorescence images were acquired at wavelengths of 425-475 nm (blue), 500-550 nm (green), 570-620 nm (red), and 650-750 nm (infrared). The images were analyzed for brightness using ImageJ.

[0025] In tissue sections that were not exposed to light, granular fluorescence, such as that of lipofuscin granules, was clearly observed, particularly at infrared wavelengths. The granular fluorescence gradually weakened after the start of light exposure and disappeared after 30 minutes. Specifically, fluorescent tissue sections of unstained mouse cerebral cortex were observed at wavelengths of 425-475 nm (blue), 500-550 nm (green), 570-620 nm (red), and 650-750 nm (infrared). Granular signals were observed at all wavelengths before light exposure, but nearly disappeared within 15 minutes of light exposure, and no granular signals were observed after 30 minutes. Furthermore, not only the granular fluorescence but also the autofluorescence observed throughout the tissue section was quenched by light exposure, and after 30 minutes, all wavelengths except blue were quenched to less than 50% of their initial intensity. Blue fluorescence faded by approximately 20% from the initial intensity (Figure 5-1). Similarly, the maximum fluorescence intensity in the sections also decreased significantly, reaching approximately 20% of the initial level at wavelengths other than blue after 30 minutes (Figure 5-2). [Explanation of symbols]

[0026] 10 Sample placement unit 12 Irradiation unit 101 Fan 102 Heat sink 103 Flat plate 104 Ribs 105 Thermometer probe insertion port 106 White LED lighting 107 Sample placement recess 108 Second Plate 109 Second Heat Sink 110 Second Fan

Claims

1. An apparatus for quenching autofluorescence of a sample, comprising a sample placement unit and an irradiation unit placed on the sample placement unit, the sample placement unit includes a flat plate, one or more sample placement recesses provided on the flat plate for placing samples therein, and a cooling means for cooling the sample placement recesses; The illumination unit includes one or more white LED lights, each of which is disposed above each of the sample placement recesses. Autofluorescence quencher.

2. 2. The autofluorescence quenching device according to claim 1, wherein the cooling means is a heat sink and / or a cooling fan provided below the flat plate.

3. 3. The autofluorescence quenching device according to claim 1, wherein the illumination unit comprises a second flat plate, and the white LED illumination is disposed on the second flat plate.

4. 4. The autofluorescence quenching device according to claim 3, wherein the irradiation unit further comprises a second cooling means.

5. 5. The autofluorescence quenching device according to claim 4, wherein the second cooling means is a heat sink and / or a cooling fan provided on the second flat plate.

6. 6. The autofluorescence quenching device according to claim 3, wherein the irradiation unit is placed on the sample placement unit via a rib, and the rib provides a space between the flat plate and the second flat plate.

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