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Optical Clearing Using SeeDB
SeeDB 光透明法   

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Abstract

We describe a water-based optical clearing agent, SeeDB (See Deep Brain), which clears fixed brain samples in a few days without quenching many types of fluorescent dyes, including fluorescent proteins and lipophilic neuronal tracers. SeeDB is a saturated solution of fructose (80.2% w/w) in water with 0.5% α-thioglycerol. In standard SeeDB optical clearing procedure, we treat paraformaldehyde-fixed embryo and brain samples with increasing concentrations of aqueous fructose solutions, and finally equilibrate them in SeeDB. The entire procedure takes approximately three days. Unlike previous methods, this method maintains a constant sample volume during the clearing procedure, an important factor to keep cellular morphology intact. After optical clearing, we can reach > 1,000 μm under confocal microscopy. When combined with two-photon microscopy, SeeDB allows us to image fixed mouse brains at millimeters-scale level. This method facilitates comprehensive and quantitative analyses for understanding neuronal circuitry, both in the adult and developing mouse brain. A SeeDB variant (SeeDB37) and optimized procedures (SeeDBp and SeeDB37ht protocols) are also supplied for specific requirements.

Keywords: Tissue clearing(组织清除), Fluorescence imaging(荧光成像), Connectome(连接体), SeeDB(SeeDB), Fructose(果糖)

Materials and Reagents

  1. Phosphate-buffered saline (PBS)
  2. 4% paraformaldehyde (PFA) in PBS
  3. D(-)-Fructose (≥99%) (e.g., Sigma-Aldrich, catalog number: F0127-500G )
  4. α-thioglycerol (≥95%) (e.g., Sigma-Aldrich, catalog number: M1753-100ML )
  5. 2,2’-thiodiethanol  (≥99%) (e.g., Sigma-Aldrich, catalog number: 166782-500G ) (optional)
  6. Glycerol (≥99%) (e.g., Sigma-Aldrich, catalog number: G9012-500ML ) (optional)
  7. Low melting point agarose (e.g., Life Technologies, catalog number: 16520 -100) (optional)
  8. SeeDB (see Recipes)
  9. SeeDBp (see Recipes)
  10. Immersion solution (for commercial objective lenses) (see Recipes)
  11. Immersion solution (for customized objective lens for SeeDB) (see Recipes)

Equipment

  1. 50 ml conical centrifuge tube (e.g., BD Biosciences, Falcon®) (for whole- and hemi-brain samples)
  2. Culture dish (30 or 60 mm diameter) (e.g., BD Biosciences, Falcon®) (for slice preparation and fragile samples)
  3. Overhead tube rotator (recommended for clearing large samples) (Figure 1A)
  4. Seesaw shaker (for fragile samples) (Figure 1B)


    Figure 1. Equipment used for optical clearing. For efficient optical clearing, it is important to shake samples in fructose solutions. We recommend using 50 ml conical centrifuge tube and overhead rotator for clearing large samples (e.g., whole- or hemi-brain samples). Fragile samples (e.g., brain slices) can be cleared in culture dishes and seesaw shaker. A. Overhead tube rotator, B. Seesaw shaker

  5. Air incubator (optional, for SeeDB37 and SeeDB37ht protocols)
  6. Coverslips (24 mm x 60 mm for small preparation and slices; 50 mm x 70 mm for whole-mount samples) (e.g., Matsunami Glass, catalog number: C050701 )
  7. Handmade glass bottom Petri dish (100 mm diameter) or a commercial glass bottom dish (MatTek, catalog number: P100G-1.5-30-F )


    Figure 2. Imaging chambers for fluorescence microscopy. A. An imaging chamber for inverted microscopes. B. An imaging chamber for upright microscopes with short working distance objective lenses. C. An imaging chamber for upright microscopes with long working distance objective lenses. We use glass bottom Petri dish to keep immersion solutions.

  8. Silicone rubber sheet (optimal thickness should be chosen. For example, 6-mm-thick silicone sheet is recommended for 6-mm thick adult mouse brain.) (e.g., TOGAWA RUBBER, catalog number: K-125 )
    Note: Silicone rubber sheets adhere to the coverslips and Petri dishes without any adhesives.
  9. Thermo plate (optional, only for samples cleared with SeeDB37 or SeeDB37ht) (e.g., Tokai Hit)
  10. Fluorescence microscope
    Confocal microscope (for imaging up to 1-2 mm depth)
    Multiphoton microscope (recommended for deeper imaging)
  11. Objective lenses (examples)
    10x air (NA=0.4, WD=3.1 mm) (OLYMPUS, model: UPLSAPO10X2 )
    10x water immersion (NA=0.3, WD=3.5 mm) (OLYMPUS, model: UMPLFLN10xW )
    25x water immersion (NA=1.05, WD=2 mm) (OLYMPUS, model: XLPLN25XWMP )
    25x scale immersion (NA=1.0, WD=4 mm) (OLYMPUS, model: XLPLN25SVMP )
    Water-immersion lenses perform much better than air-immersion objective lenses because spherical aberrations are smaller (Figure 5). In the two-photon microscopy, we also use a custom-made objective lens that performs best under the refractive index of SeeDB (~1.49). Please contact Olympus for its availability.

Procedure

Part I. Choice of protocols

  1. Standard SeeDB and SeeDB37: brain slice, mouse embryo, young mouse whole-brain, and adult mouse hemi-brain
  2. SeeDBp: embryonic and neonatal mouse brain
  3. SeeDB37ht: large samples, such as adult mouse whole-brain


    Figure 3. Timing of SeeDB protocols

    1. SeeDB (standard)
      1. Fix the sample in 4% PFA at 4 °C with gentle shaking overnight.
      2. Wash the sample in PBS three times (10 min each).
      3. (Optional step for fragile samples) Embed the sample in 1% low melting point agarose gel in PBS with desired orientation and then trim away extra portion to minimize the sample size. The surface of the sample should be close to the surface of agarose gel, because the working distance of commercially available objective lens is limited. Agarose embedding should not be used for large tissues, because agarose embedding reduces the penetration of SeeDB into the samples.
      4. Incubate the sample in ~20 ml of 20% (w/v) fructose solution in 50 ml conical tube, and then place the conical tube on a tube rotator (~4 rpm) or a seesaw shaker (~17 rpm) for 4-8 h, respectively. A small piece of sample (e.g., slices) requires less time for optical clearing. Incubation should be performed at 25-37 °C.
      5. Incubate the sample in 40% (w/v) fructose for 4-8 h as above.
      6. Incubate the sample in 60% (w/v) fructose for 4-8 h. (Samples may no longer sink in 60% or higher concentrations of fructose.)
      7. Incubate the sample in 80% (w/v) fructose for 12 h. 
      8. Incubate the sample in 100% (w/v) fructose for 12 h.
      9. Incubate the sample in ~20 ml SeeDB for 24 h. The incubation time can be extended up to 48 h.
      10. The transparency can be evaluated by eye at this stage (Figure 4). If the sample is successfully cleared, the adult brain sample should look amber under a light source.
      11. If the clarity is still not enough, incubate the sample further in ~20 ml SeeDB37 solution at 37 °C (in an air incubator) with gentle rotation for 24 h. We recommend this step for adult brain samples.
    2. SeeDBp
      Incubation schedule for SeeDBp is the same as the standard SeeDB protocol. To prevent sample expansion, 20% ,40%, 60%, and 80% fructose solutions are made in 0.1x PBS instead of distilled water.
    3. SeeDB37ht
      Note: Fluorescent proteins may be partially quenched. Some sample expansion may occur.
      1. Fix the sample in 4% PFA at 4 °C with gentle shaking overnight.
      2. Wash the sample in PBS three times (10 min each).
      3. Incubate the sample in 20% (w/v) fructose at 50 °C for 2-4 h. To prevent sample expansion, the incubation time should not exceed 4 h.
      4. Incubate the sample in 40% (w/v) fructose at 50 °C for 2-4 h.
      5. Incubate the sample in 60% (w/v) fructose at 50 °C for 2-4 h.
      6. Incubate the sample in 80% (w/v) fructose at 50 °C for 2-4 h.
      7. Incubate the sample in 100% (w/v) fructose at 50 °C for 12 h.
      8. Incubate the sample in SeeDB at 50 °C for 24 h.
      9. Incubate the sample in SeeDB37 at 50 °C for 24 h. Transfer the sample to 37 °C for imaging. Extension of incubation at 50 °C is not recommended.

Part II. Choice of objective lenses and imaging
Because refractive index of SeeDB is 1.49, it is important to choose appropriate objective lenses to minimize spherical aberration. In the two-photon microscopy, index-matched and long working distance customized objective lens is most ideal to obtain high-resolution images at millimeter-scale. We used custom-made objective lens from Olympus (it will be commercialized from Olympus). Commercial water or Scale immersion objective lens can be used for imaging up to 2-4 mm depth. In the confocal microscopy, a water-immersion objective lens (refractive index 1.33) works better than an air-immersion objective lens (refractive index 1.0) (Figure 5). Glycerol/Oil immersion lenses are also suitable. Customized objective lens is not necessary for confocal imaging; we did not see obvious differences in resolution within 1 mm depth. When using an air or water-immersion lens, the imaging depth should be calibrated because an apparent axial scale is shortened in the SeeDB solution due to its high refractive index (refractive index 1.49).

  1. Imaging with upright microscopes
    1. Prepare an appropriate imaging chamber (Figure 2B-C) for your microscope.
    2. Fill in the imaging chamber with SeeDB or SeeDB37. To prevent subtle index mismatch between media and sample, we recommend using SeeDB or SeeDB37 that was equilibrated with the sample, rather than freshly prepared solutions.
    3. Put the sample into the imaging chamber and then place a handmade glass bottom Petri dish (or a coverslip) carefully above the chamber. Air bubble should not remain in the chamber. When samples are cleared by SeeDB37, the chamber should be placed on a heat pad to keep the temperature at 37 °C.
    4. (Optional for an upright microscope) Pour immersion solution onto the handmade glass bottom Petri dish. SeeDB should not be used for immersion; its viscosity causes an uneven refraction index distribution due to evaporation of water from the surface of SeeDB during imaging and can impair image quality.
  2. Imaging with inverted microscopes
    1. Transfer the sample and SeeDB to a custom-made imaging chamber (Figure 2A) or a commercial glass-bottomed dish/chamber (35 mm diameter).


Anticipated Results

Figure 4. Transmittance images of neonatal, P21, and adult mouse brain samples. Transparency can be evaluated by eye under the light source. Typically, adult samples are more difficult to clear than young samples.

Figure 5. Confocal imaging of adult brain slices (Thy1-YFP-H mouse). Water-immersion objective lenses work better than air lenses because of smaller spherical aberration in SeeDB. The maximum imaging depth possible is ~500 μm at spine resolution, ~1 mm at fiber resolution, and ~2 mm at cellular resolution. In the two-photon microscopy, the maximum imaging depth possible with commercial 25x objective lenses is ~1 mm at spine resolution and 3-4 mm at fiber resolution. When a commercial water- or air-immersion objective lens is used for imaging cleared samples, depth (z) needs to be calibrated. In order to obtain the correct z position in the sample, the real depth is calculated by multiplying the depth by nSeeDB and then dividing by nobjective, where n represents refractive index. nSeeDB = 1.49; nSeeDB37 = 1.50; nair = 1.0; nH2O = 1.33.

Recipes

  1. Preparation of SeeDB
    Dissolve D(-)-fructose completely in distilled water or 0.1x PBS at 65 °C. We recommend using 50 ml conical centrifuge tube. After cooling to 25 °C or 37 °C, add α-thioglycerol to give a final concentration of 0.5% to prevent Maillard reaction.
    Notes:
    a. 20-100% fructose solutions are weight/volume and SeeDB/SeeDB37 are prepared on the basis of percent weight/weight. SeeDB should be freshly prepared.
    b. Do not leave SeeDB solutions at 65 °C too long (> 5 h), because fructose will gradually caramelize.

    Composition
    Fructose
    Solvent
    α-thioglycerol
    20% w/v
    4 g
    add distilled water to make a total volume of 20 ml
    100 μl
    40% w/v
    8 g
    60% w/v
    12 g
    80% w/v
    16 g
    100% w/v
    20 g
    SeeDB
    20.25 g
    add 5 ml distilled water
    SeeDB37
    27 g

  2. Preparation of SeeDBp

    Composition
    Fructose
    Solvent
    α-thioglycerol
    20% w/v
    4 g
    add 0.1x PBS to make a total volume of 20 ml
    100 μl


    40% w/v
    8 g
    60% w/v
    12 g
    80% w/v
    16 g
    100% w/v
    20 g
    add distilled water to 20 ml
    SeeDB
    20.25 g
    add 5 ml distilled water
    SeeDB37
    27 g

  3. Immersion solution (for commercial objective lenses)
    Water (for water-immersion lens)
    30% glycerol (v/v, in distilled water; for Scale-immersion lens)
  4. Immersion solution (for customized objective lens for SeeDB)
    80% 2,2’-thiodiethanol – 20% H2O (v/v; for SeeDB)
    90% 2,2’-thiodiethanol – 10% H2O (v/v; for SeeDB37)

Troubleshooting

  1. Poor transparency
    Because SeeDB is viscous, it is important to ensure efficient penetration of the solution into the tissues. If possible, trim away unnecessary portion of your sample to increase the penetration. Remove skin and skull, which are typically very difficult to clear. If you need to clear large samples (e.g., whole brains of adult mice), try SeeDB37ht protocol. Shaking of samples, step-wise soaking, timing, and temperature are all important for successful optical clearing.
  2. Autofluorescence
    Prolonged incubation in SeeDB will accumulate autofluorescence even with 0.5% α-thioglycerol. It is ideal to image samples soon after the clearing (within a few days). For long-term storage, samples should be recovered in PBS following exactly the reverse of the clearing process. No detergents and salts should be included in SeeDB (except for SeeDBp). PFA should be freshly prepared to minimize autofluorescence.
  3. High temperature protocol (SeeDB37ht)
    Incubation of the sample at higher temperature may result in mild sample expansion. To minimize the sample expansion, incubation time for 20-80% fructose should be minimized. In the SeeDB37ht protocol, some fluorescent protein may be partially quenched.
  4. In utero electroporation
    Do not use FastGreen dye, because it will react with fructose and result in strong autofluorescence. We instead recommend 0.05% AlexaFluor 647-dextran when you need to visualize DNA solution.
  5. Antibody staining
    SeeDB can also be used to clear samples stained with antibodies, although penetration of antibodies is typically limited to 100-250 μm even after permeabilization step with Triton-X100. Because optical clearing with SeeDB is reversible, samples cleared with SeeDB can be restored in PBS and subsequently analyzed by immunohistochemistry in sections.

Acknowledgments

This protocol was adapted from our original publication of the protocol (Ke et al., 2013). We thank J.R. Sanes for providing mouse strains. This work was supported by grants from the PRESTO program of the Japan Science and Technology Agency, the Sumitomo Foundation, the Nakajima Foundation, the Mitsubishi Foundation, the Strategic Programs for R&D (President's Discretionary Fund) of RIKEN, and the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (KAKENHI 23680038). The imaging experiments were supported by the RIKEN Center for Developmental Biology Imaging Facility. Animal experiments were supported by the Laboratory for Animal Resources and Genetic Engineering at the RIKEN Center for Developmental Biology.

References

  1. Ke, M. T., Fujimoto, S. and Imai, T. (2013). SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Nat Neurosci 16(8): 1154-1161.

    Note: SeeDB Resources (https://sites.google.com/site/seedbresources/) provides updated information from the authors.


This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

简介

我们描述了一种水基光学清除剂,SeeDB(参见深脑),其在几天内清除固定的脑样品,而不淬灭许多类型的荧光染料,包括荧光蛋白和亲脂性神经元示踪剂。 SeeDB是果糖(80.2%w/w)在含有0.5%α-硫代甘油的水中的饱和溶液。在标准的SeeDB光学清除程序中,我们用增加浓度的果糖水溶液处理多聚甲醛固定的胚胎和脑样品,并且最后在SeeDB中平衡它们。整个过程大约需要三天。与以前的方法不同,该方法在清除过程中保持恒定的样品体积,这是保持细胞形态完整的重要因素。光学清除后,我们可以达到>在共聚焦显微镜下为1,000μm。当与双光子显微镜结合时,SeeDB允许我们以毫米级别水平成像固定的小鼠大脑。这种方法有助于全面和定量分析理解神经元电路,在成人和发展中国家的小鼠大脑。还提供了SeeDB变体(参见DB37)和优化的程序(见DBp和SeeDB37ht协议)以满足特定要求。

关键字:组织清除, 荧光成像, 连接体, SeeDB, 果糖

材料和试剂

  1. 磷酸盐缓冲盐水(PBS)
  2. 4%多聚甲醛(PFA)在PBS中的溶液
  3. D( - ) - 果糖(≥99%)(例如,Sigma-Aldrich,目录号:F0127-500G)
  4. α-硫代甘油(≥95%)(例如,Sigma-Aldrich,目录号:M1753-100ML)
  5. (≥99%)(如,Sigma-Aldrich,目录号:166782-500G)(可选)
  6. 甘油(≥99%)(如,Sigma-Aldrich,目录号:G9012-500ML)(可选)
  7. 低熔点琼脂糖(例如,Life Technologies,目录号:16520-100)(可选)
  8. SeeDB(参见配方)
  9. 参见dbp(参见配方)
  10. 浸没溶液(用于商业物镜)(参见配方)
  11. 浸没溶液(用于SeeDB的定制物镜)(参见配方)

设备

  1. (用于全脑和半脑样品)的250ml锥形离心管(例如BD Biosciences,Falcon )。
  2. 培养皿(30或60mm直径)(例如,BD Biosciences,Falcon )(用于切片制备和脆性样品)
  3. 架空管旋转器(推荐用于清除大样品)(图1A)
  4. 跷板振动器(用于脆性样品)(图1B)


    图1.用于光学清洗的设备。为了有效地进行光学清洗,必须在果糖溶液中摇动样品。我们建议使用50ml圆锥形离心管和顶部旋转器清除大样本(例如全脑或半脑样品)。可以在培养皿和跷跷板振荡器中清除脆性样品(例如脑切片)。 A.架空管旋转器,B.跷跷板摇动器

  5. 空气培养箱(可选,SeeDB37和SeeDB37ht协议)
  6. 盖片(24mm×60mm用于小制备和切片; 50mm×70mm用于整装样品)(例如,Matsunami Glass,目录号:C050701)
  7. 手工玻璃底培养皿(100mm直径)或商业玻璃底培养皿(MatTek,目录号:P100G-1.5-30-F)


    图2.用于荧光显微镜的成像室。 A.用于倒置显微镜的成像室。 B.用于具有短工作距离物镜的直立显微镜的成像室。 C.用于具有长工作距离物镜的直立显微镜的成像室。我们使用玻璃底培养皿保持浸泡解决方案
  8. 硅胶橡胶片(应选择最佳厚度,例如,对于6mm厚的成年老鼠大脑推荐使用6mm厚的硅胶片)(,TOGAWA RUBBER,目录号:K- 125)
    注意:硅胶片粘附在盖玻片和培养皿上,没有任何粘合剂。
  9. 热板(可选,仅用于通过SeeDB37或SeeDB37ht清除的样品)(例如,Tokai Hit)
  10. 荧光显微镜
    共焦显微镜(用于高达1-2mm深度的成像)
    多光子显微镜(推荐用于更深的成像)
  11. 物镜(例子)
    10x空气(NA = 0.4,WD = 3.1mm)(OLYMPUS,型号:UPLSAPO10X2)
    10x水浸(NA = 0.3,WD = 3.5mm)(OLYMPUS,型号:UMPLFLN10xW)
    25x水浸(NA = 1.05,WD = 2mm)(OLYMPUS,型号:XLPLN25XWMP)
    25x标度浸没(NA = 1.0,WD = 4mm)(OLYMPUS,型号:XLPLN25SVMP)
    水浸透镜的性能比空气浸没物镜好得多,因为球面像差更小(图5)。 在双光子显微镜中,我们还使用定制的物镜在SeeDB(〜1.49)的折射率下表现最好。 请联系Olympus了解其可用性。

程序

第一部分

  1. 标准SeeDB和SeeDB37:脑切片,小鼠胚胎,幼小鼠全脑和成年小鼠半脑
  2. SeeDBp:胚胎和新生小鼠脑
  3. SeeDB37ht:大样本,如成年小鼠全脑


    图3. SeeDB协议的时间

    1. SeeDB(标准)
      1. 固定样品在4%PFA在4℃,轻轻摇动过夜。
      2. 在PBS中洗涤样品三次(每次10分钟)。
      3. (脆性样品的任选步骤)将样品包埋在具有所需取向的PBS中的1%低熔点琼脂糖凝胶中,然后修剪掉多余部分以使样品尺寸最小化。样品的表面应该接近琼脂糖凝胶的表面,因为市售的物镜的工作距离有限。琼脂糖包埋不应用于大组织,因为琼脂糖包埋降低了SeeDB渗透到样品中。
      4. 将样品在50ml锥形管中的约20ml 20%(w/v)果糖溶液中孵育,然后将锥形管置于管旋转器(〜4rpm)或跷跷板摇动器(〜17rpm) 8小时。 一小片样品(例如,切片)需要较少的时间用于光学清除。孵育应在25-37℃进行。
      5. 如上所述将样品在40%(w/v)果糖中孵育4-8小时。
      6. 孵育样品在60%(w/v)果糖4-8小时。 (样品可能不再在60%或更高浓度的果糖下沉淀。)
      7. 将样品在80%(w/v)果糖中孵育12小时。
      8. 孵育样品在100%(w/v)果糖12小时。
      9. 孵育样品在〜20ml,SeeDB 24小时。 孵育时间可延长至48小时。
      10. 在该阶段可以通过眼睛评价透明度(图4)。 如果样品成功清除,成人脑样品应在光源下呈琥珀色。
      11. 如果澄清度仍然不够,在37℃下(在空气培养箱中),在约20ml的SeeDB37溶液中温育样品,轻轻旋转24小时。 我们建议成人脑样本的这一步。
    2. 参见dbp
      SeeDBp的孵化计划与标准SeeDB协议相同。 为了防止样品膨胀,在0.1x PBS而不是蒸馏水中制备20%,40%,60%和80%的果糖溶液。
    3. 参见db37ht
      注意:荧光蛋白可能被部分淬灭。 可能会发生一些样本扩展。
      1. 固定样品在4%PFA在4℃,轻轻摇动过夜。
      2. 在PBS中洗涤样品三次(每次10分钟)。
      3. 孵育样品在20%(w/v)果糖50℃下2-4小时。 为了防止样品扩增,孵育时间不应超过4小时。
      4. 将样品在40%(w/v)果糖中于50℃孵育2-4小时。
      5. 孵育样品在60%(w/v)果糖50℃下2-4小时。
      6. 孵育样品在80%(w/v)果糖在50℃下2-4小时。
      7. 将样品在100%(w/v)果糖中于50℃孵育12小时。
      8. 将样品在SeeDB中在50℃孵育24小时。
      9. 将样品在SeeDB37中在50℃孵育24小时。 将样品转移至37℃进行成像。 不推荐在50°C下延长孵育时间。

第二部分。 选择物镜和成像
因为SeeDB的折射率为1.49,所以重要的是选择适当的物镜以使球面像差最小化。在双光子显微镜中,折射率匹配和长工作距离定制物镜对于以毫米级获得高分辨率图像是最理想的。我们使用奥林巴斯的定制物镜(它将从奥林巴斯商业化)。商用水或标准浸入物镜可用于高达2-4mm深度的成像。在共聚焦显微镜中,浸水物镜(折射率1.33)比空气浸没物镜(折射率1.0)(图5)更好。甘油/油浸镜头也是合适的。定制物镜对于共焦成像不是必需的;我们没有看到1毫米深度内分辨率的明显差异。当使用空气或水浸透镜时,成像深度应当被校准,因为SeeDB溶液中的表观轴向尺度由于其高折射率(折射率1.49)而缩短。

  1. 直立显微镜成像
    1. 为您的显微镜准备一个适当的成像室(图2B-C)。
    2. 用SeeDB或SeeDB37填充成像室。为了防止介质和样品之间微妙的指数不匹配,我们建议使用与样品平衡的SeeDB或SeeDB37,而不是新鲜制备的溶液。
    3. 将样品放入成像室,然后放置手工玻璃底培养皿(或盖玻片)小心地在室上方。气泡不应留在腔室中。清除样本时 通过SeeDB37,室应该放置在热垫上以保持温度在37℃。
    4. (直立显微镜可选)将浸泡液倒入手工制作的玻璃底培养皿中。 SeeDB不应该用于浸没; 其粘度由于成像期间水从SeeDB表面的蒸发而导致不均匀的折射率分布,并且可能削弱图像质量。
  2. 倒置显微镜成像
    1. 将样品和SeeDB转移到定制的成像室(图2A)或商业玻璃底的皿/室(35mm直径)。


预期结果

图4.新生儿,P21和成年小鼠脑样品的透射图像。透明度可以在光源下用眼睛评估。通常,成人样品比年轻样品更难以清除

图5.成体脑切片( 1-YFP-H 小鼠)的共聚焦成像。浸水物镜比空气透镜工作更好,因为SeeDB中的球面像差更小。在脊柱分辨率下,最大成像深度为〜500μm,在光纤分辨率下为〜1mm,在蜂窝分辨率下为〜2mm。在双光子显微镜中,商用25x物镜的最大成像深度在脊柱分辨率为〜1mm,在光纤分辨率为3-4mm。当商用水或空气浸没物镜用于对清除的样本成像时,需要校准深度(z)。为了在样本中获得正确的z位置,通过将深度乘以n subSub,然后除以n sub目标来计算真实深度,其中n表示折射率。 n SeeDB = 1.49; n SeeDB37 = 1.50; n空气= 1.0; n H 2 O = 1.33。

食谱

  1. 准备SeeDB
    在65℃下将D( - ) - 果糖完全溶解在蒸馏水或0.1x PBS中。 我们建议使用50ml锥形离心管。 冷却至25℃或37℃后,加入α-硫代甘油至终浓度为0.5%以防止美拉德反应。
    注意:
    a。 20-100%果糖溶液的重量/体积,并且基于重量/重量百分比制备了SeeDB/SeeDB37。 SeeDB应该是新鲜准备的。
    b。 不要将SeeDB溶液在65°C下保持太长时间(> 5小时),因为果糖会逐渐变焦。

    组合
    果糖
    溶剂
    α-硫代甘油
    20%w/v
    4克
    加入蒸馏水,使总体积为20ml 100微升
    40%w/v
    8克
    60%w/v
    12克
    80%w/v
    16克
    100%w/v
    20克
    SeeDB
    20.25克
    加入5ml蒸馏水
    参见db37
    27克

  2. Seedbp的准备

    组合
    果糖
    溶剂
    α-硫代甘油
    20%w/v
    4克
    加入0.1x PBS使总体积为20ml ml
    100μl


    40%w/v
    8克
    60%w/v
    12克
    80%w/v
    16克
    100%w/v
    20克
    加蒸馏水至20ml
    SeeDB
    20.25克
    加入5ml蒸馏水
    参见db37
    27克

  3. 浸没溶液(用于商业物镜)
    水(用于浸水透镜)
    30%甘油(v/v,在蒸馏水中;用于水垢镜)
  4. 浸没溶液(用于SeeDB的定制物镜)
    80%2,2'-硫代二乙醇-20%H 2 O(v/v;对于SeeDB)
    90%2,2'-硫代二乙醇-10%H 2 O(v/v;对于SeeDB37)

故障排除

  1. 透明度差
    因为SeeDB是粘性的,所以重要的是确保溶液有效地渗透到组织中。如果可能,修剪掉样品的不必要部分以增加渗透。去除皮肤和头骨,这通常是很难清除。如果你需要清除大样本(例如,成年小鼠的整个大脑),请尝试SeeDB37ht协议。样品的摇动,逐步浸泡,定时和温度对于成功的光学清除都是重要的
  2. 自发荧光
    在SeeDB的长时间孵育将积累自身荧光,甚至与0.5%α-硫代甘油。它是理想的清除后(几天内)图像样品。对于长期储存,样品应在PBS中回收 恰恰与结算过程相反。在SeeDB中不应包括去污剂和盐(除了SeeDBp之外)。 PFA应该新鲜制备以最小化自发荧光。
  3. 高温方案(见DB37ht)
    在较高温度下孵育样品可导致温和的样品膨胀。为了使样品膨胀最小化,应当使20-80%果糖的温育时间最小化。在SeeDB37ht协议中,一些荧光蛋白可能被部分淬灭
  4. 在子宫内电穿孔
    不要使用FastGreen染料,因为它会与果糖反应,导致强烈的自发荧光。当您需要可视化DNA溶液时,我们建议使用0.05%的AlexaFluor 647葡聚糖
  5. 抗体染色
    SeeDB也可用于清除用抗体染色的样品,尽管抗体的穿透通常限于100-250μm,甚至在使用Triton-X100的透化步骤后。因为用SeeDB的光学清除是可逆的,用SeeDB清除的样品可以在PBS中恢复,随后通过免疫组织化学在部分中进行分析。

致谢

该协议改编自我们最初的方案出版物(Ke等人,2013)。我们感谢J.R.Sanes提供小鼠品系。这项工作得到了日本科学技术局PRESTO计划,住友基金会,中岛基金会,三菱基金会,RIKEN研究与发展战略计划(总裁独立基金)和教育部的资助,文化,体育,科学技术(MEXT)(KAKENHI 23680038)。成像实验由RIKEN发育生物学成像设备中心支持。动物实验由RIKEN发育生物学中心的动物资源和基因工程实验室支持。

参考文献

  1. Ke,M.T.,Fujimoto,S.and Imai,T。(2013)。 SeeDB:一种用于神经元回路重建的简单且保留形态的光学清除剂。 em> Nat Neurosci 16(8):1154-1161。

    注意:SeeD​​B资源(https://sites.google.com/site/seedbresources/)提供了来自作者的更新信息。


本项作品根据 Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License 授权 。

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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Ke, M., Fujimoto, S. and Imai, T. (2014). Optical Clearing Using SeeDB. Bio-protocol 4(3): e1042. DOI: 10.21769/BioProtoc.1042.
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Anirudh Venkatesh
Bioplastics Org
Where can I find the SeeDB recipes?
5/10/2015 7:31:08 AM Reply
Bio-protocol Editorial Team
bio-protocol.org

Hi, Anirudh,

Please find the SeeDB recipes in Recipes section of the protocol above.

--Bio-protocol team



5/25/2015 12:43:50 PM